Genetically modified non-human animal with human or chimeric PD-L1

ABSTRACT

This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) programmed death-ligand 1 (PD-L1, PDL1, or B7-H1), and methods of use thereof. In one aspect, the disclosure relates to genetically-modified, non-human animals whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric programmed death-ligand 1 (PD-L1).

CLAIM OF PRIORITY

This application is a continuation of and claims priority to U.S.application Ser. No. 16/329,376, filed Feb. 28, 2019, which is a 371application of and claims priority to international Application No.PCT/CN2017/099574, filed Aug. 30, 2017, which claims the benefit ofChinese Patent Application App. No. 201610775382.4, filed on Aug. 31,2016, and Chinese Patent App. No. 201710757022.6, filed on Aug. 29,2017. The entire contents of the foregoing are incorporated herein byreference.

TECHNICAL FIELD

This disclosure relates to genetically modified animal expressing humanor chimeric (e.g., humanized) programmed death-ligand 1 (PD-L1, PDL1, orB7-H1), and methods of use thereof.

BACKGROUND

Cancer is currently one of the diseases that have the highest humanmortality. According to the World Health Organization statistical data,in 2012 the number of global cancer incidence and death cases reached 14million and 8.2 million, respectively. In China, the newly diagnosedcancer cases are 3.07 million, and the death toll is 2.2 million.

In recent years, antibody drug development for immunological checkpointsis considered to be a potential target for the treatment of varioustypes of cancers. The traditional drug research and developmenttypically use in vitro screening approaches. However, these screeningapproaches cannot provide the body environment (such as tumormicroenvironment, stromal cells, extracellular matrix components andimmune cell interaction, etc.), resulting in a higher rate of failure indrug development. In addition, in view of the differences between humansand animals, the test results obtained from the use of conventionalexperimental animals for in vivo pharmacological test may not be able toreflect the real disease state and the identification and interaction atthe targeting sites, resulting in that the results in many clinicaltrials are significantly different from the animal experimental results.Therefore, the development of humanized animal models that are suitablefor human antibody screening and evaluation will significantly improvethe efficiency of new drug development and reduce the costs for drugresearch and development.

SUMMARY

This disclosure is related to PD-L1 humanized animal model. The animalmodel can express human PD-L1 or chimeric PD-L1 (e.g., humanized PD-L1)protein in its body. It can be used in the studies on the function ofPD-L1 gene, and can be used in the screening and evaluation ofanti-human PD-L1 antibodies. In addition, the animal models prepared bythe methods described herein can be used in drug screening,pharmacodynamics studies, treatments for immune-related diseases, andcancer therapy for human PD-L1 target sites; in addition, they can beused to facilitate the development and design of new drugs, and savetime and cost. In summary, this disclosure provides a powerful tool forstudying the function of PD-L1 protein and screening for cancer drugs.

Furthermore, the disclosure also provides PD-L1 gene knockout mice.Moreover, the mice described in the present disclosure can be mated withthe mice containing other human or chimeric genes (e.g., chimeric PD-1or other immunomodulatory factors), so as to obtain a mouse having ahuman or chimeric protein at both alleles of the endogenous gene. Themice can also, e.g., be used for screening antibodies in the case of acombined use of drugs, as well as evaluating the efficacy of thecombination therapy.

In one aspect, the disclosure relates to genetically-modified, non-humananimals whose genome comprises at least one chromosome comprising asequence encoding a human or chimeric programmed death-ligand 1 (PD-L1).In some embodiments, the sequence encoding the human or chimeric PD-L1is operably linked to an endogenous regulatory element at the endogenousPD-L1 gene locus in the at least one chromosome. In some embodiments,the sequence encoding a human or chimeric PD-L1 comprises a sequenceencoding an amino acid sequence that is at least 70%, 75%, 80%, 85%,90%, 95%, 99%, or 100% identical to human PD-L1 (NP 054862.1) (SEQ IDNO: 29). In some embodiments, the sequence encoding a human or chimericPD-L1 comprises a sequence encoding an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:33. In some embodiments, the sequence encoding a human or chimeric PD-L1comprises a sequence encoding an amino acid sequence that corresponds toamino acids 21-128 of SEQ ID NO: 29. In some embodiments, the animal isa mammal, e.g., a monkey, a rodent or a mouse. In some embodiments, theanimal is a C57BL/6 mouse. In some embodiments, the animal does notexpress endogenous PD-L1. In some embodiments, the animal has one ormore cells expressing human or chimeric PD-L1. In some embodiments, theanimal has one or more cells expressing human or chimeric PD-L1, and theexpressed human or chimeric PD-L1 can bind to human PD-1 anddownregulate immune response in the animal. In some embodiments, theanimal has one or more cells expressing human or chimeric PD-L1, and theexpressed human or chimeric PD-L1 can bind to endogenous PD-1 anddownregulate immune response in the animal.

In another aspect, the disclosure relates to genetically-modified,non-human animals, wherein the genome of the animal comprises areplacement, at an endogenous PD-L1 gene locus, of a sequence encoding aregion of endogenous PD-L1 with a sequence encoding a correspondingregion of human PD-L1 In some embodiments, the sequence encoding thecorresponding region of human PD-L1 is operably linked to an endogenousregulatory element at the endogenous PD-L1 locus, and one or more cellsof the animal expresses a chimeric PD-L1. In some embodiments, theanimal does not express endogenous PD-L1. In some embodiments, theregion of endogenous PD-L1 is the extracellular region of PD-L1. In someembodiments, the animal has one or more cells expressing a chimericPD-L1 having an extracellular region, a transmembrane region, and acytoplasmic region, wherein the extracellular region comprises asequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identicalto the extracellular region of human PD-L1. In some embodiments, theextracellular region of the chimeric PD-L1 has a sequence that has atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 contiguous amino acidsthat are identical to a contiguous sequence present in the extracellularregion of human PD-L1. In some embodiments, the animal is a mouse, andthe sequence encoding the region of endogenous PD-L1 is Exon 1, Exon 2,Exon 3, Exon 4, Exon 5, Exon 6, and/or Exon 7 of the endogenous mousePD-L1 gene. In some embodiments, the animal is heterozygous with respectto the replacement at the endogenous PD-L1 gene locus. In someembodiments, the animal is homozygous with respect to the replacement atthe endogenous PD-L1 gene locus.

In another aspect, the disclosure relates to methods for making agenetically-modified, non-human animal, including: replacing in at leastone cell of the animal, at an endogenous PD-L1 gene locus, a sequenceencoding a region of an endogenous PD-L1 with a sequence encoding acorresponding region of human PD-L1 In some embodiments, the sequenceencoding the corresponding region of human PD-L1 comprises exon 1, exon2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of a human PD-L1 gene.In some embodiments, the sequence encoding the corresponding region ofPD-L1 comprises a portion of exon 3 of a human PD-L1 gene. In someembodiments, the sequence encoding the corresponding region of humanPD-L1 encodes amino acids 21-128 of SEQ ID NO: 29. In some embodiments,the region is located within the extracellular region of PD-L1. In someembodiments, the animal is a mouse, and the sequence encoding the regionof the endogenous PD-L1 locus is Exon 3 of mouse PD-L1 gene.

In another aspect, the disclosure relates to non-human animalscomprising at least one cell comprising a nucleotide sequence encoding achimeric PD-L1 polypeptide, wherein the chimeric PD-L1 polypeptidecomprises at least 50 contiguous amino acid residues that are identicalto the corresponding contiguous amino acid sequence of a human PD-L1,wherein the animal expresses the chimeric PD-L1. In some embodiments,the chimeric PD-L1 polypeptide has at least 50 contiguous amino acidresidues that are identical to the corresponding contiguous amino acidsequence of a human PD-L1 extracellular region. In some embodiments, thechimeric PD-L1 polypeptide comprises a sequence that is at least 90%,95%, or 99% identical to amino acids 42-145 of SEQ ID NO: 33. In someembodiments, the nucleotide sequence is operably linked to an endogenousPD-L1 regulatory element of the animal. In some embodiments, thechimeric PD-L1 polypeptide comprises an endogenous PD-L1 transmembraneregion and/or an endogenous PD-L1 cytoplasmic region. In someembodiments, the nucleotide sequence is integrated to an endogenousPD-L1 gene locus of the animal. In some embodiments, the chimeric PD-L1has at least one mouse PD-L1 activity and/or at least one human PD-L1activity.

In another aspect, the disclosure relates to methods of making agenetically-modified mouse cell that expresses a chimeric PD-L1, themethod including: replacing, at an endogenous mouse PD-L1 gene locus, anucleotide sequence encoding a region of mouse PD-L1 with a nucleotidesequence encoding a corresponding region of human PD-L1, therebygenerating a genetically-modified mouse cell that includes a nucleotidesequence that encodes the chimeric PD-L1, wherein the mouse cellexpresses the chimeric PD-L1 In some embodiments, the chimeric PD-L1comprises an extracellular region of mouse PD-L1 comprising a mousesignal peptide sequence; an extracellular region of human PD-L1; atransmembrane and/or a cytoplasmic region of a mouse PD-L1. In someembodiments, the nucleotide sequence encoding the chimeric PD-L1 isoperably linked to an endogenous PD-L1 regulatory region, e.g.,promoter. In some embodiments, the animal further comprises a sequenceencoding an additional human or chimeric protein. In some embodiments,the additional human or chimeric protein is programmed cell deathprotein 1 (PD-1), TNF Receptor Superfamily Member 4 (OX40), LymphocyteActivating 3 (LAG-3), T-Cell Immunoglobulin And Mucin Domain-ContainingProtein 3 (TIM-3), CTLA-4, TNF Receptor Superfamily Member 9 (4-1BB),CD27, CD28, CD47, T-Cell Immunoreceptor With Ig And ITIM Domains(TIGIT), CD27, Glucocorticoid-Induced TNFR-Related Protein (GITR), or BAnd T Lymphocyte Associated (BTLA). In some embodiments, the animal ormouse further comprises a sequence encoding an additional human orchimeric protein. In some embodiments, the additional human or chimericprotein is programmed cell death protein 1 (PD-1), OX40, LAG-3, TIM-3,CTLA-4, 4-1BB, CD27, CD28, CD47, TIGIT, CD27, GITR, or BTLA.

In another aspect, the disclosure relates to methods of determiningeffectiveness of an anti-PD-L1 antibody for the treatment of cancer,including administering the anti-PD-L1 antibody to the animal of any oneof the embodiments disclosed herein, wherein the animal has a tumor; anddetermining the inhibitory effects of the anti-PD-L1 antibody to thetumor. In some embodiments, the animal comprises one or more immunecells (e.g., T cells) that express PD-1. In some embodiments, the tumorcomprises one or more cancer cells that are injected into the animal. Insome embodiments, determining the inhibitory effects of the anti-PD-L1antibody to the tumor involves measuring the tumor volume in the animal.In some embodiments, the tumor cells are melanoma cells, non-small celllung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells,bladder cancer cells, prostate cancer cells (e.g., metastatichormone-refractory prostate cancer), breast cancer cells, and/orurothelial carcinoma cells.

In another aspect, the disclosure relates to methods of determiningeffectiveness of an anti-PD-L1 antibody and an additional therapeuticagent for the treatment of a tumor, including administering theanti-PD-L1 antibody and the additional therapeutic agent to the animalof any one of the embodiments disclosed herein, wherein the animal has atumor; and determining the inhibitory effects on the tumor. In someembodiments, the animal further comprises a sequence encoding a human orchimeric programmed cell death protein 1 (PD-1). In some embodiments,the additional therapeutic agent is an anti-PD-1 antibody. In someembodiments, the animal comprises one or more immune cells (e.g., Tcells) that express PD-1. In some embodiments, the tumor comprises oneor more tumor cells that express PD-L1 or PD-L2. In some embodiments,the tumor is caused by injection of one or more cancer cells into theanimal. In some embodiments, determining the inhibitory effects of thetreatment involves measuring the tumor volume in the animal. In someembodiments, the tumor comprises melanoma cells, non-small cell lungcarcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, bladdercancer cells, prostate cancer cells (e.g., metastatic hormone-refractoryprostate cancer cells), breast cancer cells, and/or urothelial carcinomacells.

In another aspect, the disclosure relates to proteins comprising anamino acid sequence, wherein the amino acid sequence is one of thefollowing: (a) an amino acid sequence set forth in SEQ ID NO: 33; (b) anamino acid sequence that is at least 90% identical to SEQ ID NO: 33; (c)an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 33; (d) an amino acid sequencethat is different from the amino acid sequence set forth in SEQ ID NO:33 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and (e)an amino acid sequence that comprises a substitution, a deletion and/orinsertion of one, two, three, four, five or more amino acids to theamino acid sequence set forth in SEQ ID NO: 33. In some embodiments, thedisclosure relates to cells comprising the proteins disclosed herein. Insome embodiments, the disclosure relates to animals comprising theproteins disclosed herein.

In another aspect, the disclosure relates to nucleic acids comprising anucleotide sequence, wherein the nucleotide sequence is one of thefollowing: (a) a sequence that encodes the protein of claim 54; (2) SEQID NO: 31; (c) SEQ ID NO: 32; (d) a sequence that is at least 90%identical to SEQ ID NO: 31 or SEQ ID NO: 32; (e) a sequence that is atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 31; and (f) a sequence that is at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO: 32. In some embodiments,the disclosure relates to cells comprising the nucleic acids disclosedherein. In some embodiments, the disclosure relates to animalscomprising the nucleic acids disclosed herein.

In one aspect, the disclosure relates to a targeting vector, includinga) a DNA fragment homologous to the 5′ end of a region to be altered (5′arm), which is selected from the PD-L1 gene genomic DNAs in the lengthof 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding adonor region; and c) a second DNA fragment homologous to the 3′ end ofthe region to be altered (3′ arm), which is selected from the PD-L1 genegenomic DNAs in the length of 100 to 10,000 nucleotides.

In some embodiments, a) the DNA fragment homologous to the 5′ end of aregion to be altered (5′ arm/receptor) is selected from the nucleotidesequences that have at least 90% homology to the NCBI accession numberNC_000085.6; c) the DNA fragment homologous to the 3′ end of the regionto be altered (3′ arm/receptor) is selected from the nucleotidesequences that have at least 90% homology to the NCBI accession numberNC_000085.6.

In some embodiments, a) the DNA fragment homologous to the 5′ end of aregion to be altered (5′ arm/receptor) is selected from the nucleotidesfrom the position 29371941 to position 29373565 of the NCBI accessionnumber NC_000085.6; c) the DNA fragment homologous to the 3′ end of theregion to be altered (3′ arm/receptor) is selected from the nucleotidesfrom the position 29373890 to position 29375042 of the NCBI accessionnumber NC_000085.6.

In some embodiments, a length of the selected genomic nucleotidesequence is 1.5 kb and 1.2 kb. In some embodiments, the region to bealtered is exon 3 of PD-L1 gene.

In some embodiments, the sequence of the 5′ arm is shown in SEQ ID NO:34. In some embodiments, the sequence of the 3′ arm is shown in SEQ IDNO: 42.

In some embodiments, the targeting vector further includes a selectablegene marker.

In some embodiments, the target region is derived from human. In someembodiments, the target region is a part or entirety of the nucleotidesequence of a humanized PD-L1. In some embodiments, the nucleotidesequence is shown as one or more of the first exon, the second exon, thethird exon, the fourth exon or the fifth exon of the human PD-L1.

In some embodiments, the nucleotide sequence of the human PD-L1 encodesthe human PD-L1 protein with the NCBI accession number NP_054862.1. Insome embodiments, the target region is shown in SEQ ID NO: 37.

The disclosure also relates to a cell including the targeting vector asdescribed herein.

In another aspect, the disclosure relates to an sgRNA sequence forconstructing a humanized animal model, wherein the sgRNA sequencetargets the PD-L1 gene, the sgRNA is unique on the target sequence ofthe PD-L1 gene to be altered, and meets the sequence arrangement rule of5′-NNN (20) -NGG3′ or 5′-CCN-N(20)-3′. In some embodiments, thetargeting site of the sgRNA in the mouse PD-L1 gene is located on theexon 3 of the mouse PD-L1 gene.

In another aspect, the disclosure relates to an sgRNA sequence forconstructing a humanized animal model, wherein an upstream sequencethereof is shown as SEQ ID NO: 17, and a downstream sequence thereof isshown as SEQ ID NO: 19, and the sgRNA sequence recognizes a 5′ targetingsite.

The disclosure also relates to an sgRNA sequence for constructing ahumanized animal model, wherein an upstream sequence thereof is shown asSEQ ID NO: 18, which is obtained by adding TAGG to the 5′ end of SEQ IDNO: 17; a downstream sequence thereof is shown as SEQ ID NO: 20, whichis obtained by adding AAAC to the 5′ end of SEQ ID NO: 19, and the sgRNAsequence recognizes a 5′ targeting site.

The disclosure also relates to an sgRNA sequence for constructing ahumanized animal model, wherein an upstream sequence thereof is shown asSEQ ID NO: 21, and a downstream sequence thereof is shown as SEQ ID NO:23, and the sgRNA sequence recognizes a 3′ targeting site.

The disclosure further relates to an sgRNA sequence for constructing ahumanized animal model, wherein an upstream sequence thereof is shown asSEQ ID NO: 22, which is obtained by adding TAGG to the 5′ end of SEQ IDNO: 21; a downstream sequence thereof is shown as SEQ ID NO: 24, whichis obtained by adding AAAC to the 5′ end of SEQ ID NO: 23, and the sgRNAsequence recognizes a 3′ targeting site.

In one aspect, the disclosure relates to a construct including the sgRNAsequence as described herein.

The disclosure also relates to a cell comprising the construct asdescribed herein. In another aspect, the disclosure relates to anon-human mammalian cell, comprising the targeting vector as describedherein, and one or more in vitro transcripts of the sgRNA construct.

In some embodiments, the cell includes Cas9 mRNA or an in vitrotranscript thereof.

In some embodiments, the genes in the cell are heterozygous. In someembodiments, the genes in the cell are homozygous.

In some embodiments, the non-human mammalian cell is a mouse cell. Insome embodiments, the cell is a fertilized egg cell. In someembodiments, the cell is a germ cell. In some embodiments, the cell is ablastocyst. In some embodiments, the cell is a lymphocyte (e.g., aB-cell or a T-cell).

In another aspect, the disclosure relates to a method for establishing aPD-L1 gene humanized animal model. The methods include the steps of

(a) providing the cell, and preferably the cell is a fertilized eggcell;

(b) culturing the cell in a liquid culture medium;

(c) transplanting the cultured cell to the fallopian tube or uterus ofthe recipient female non-human mammal, allowing the cell to develop inthe uterus of the female non-human mammal;

(d) identifying the germline transmission in the offspring geneticallymodified humanized non-human mammal of the pregnant female in step (c).

In some embodiments, the establishment of a humanized animal model ofPD-L1 gene using a gene editing technique is based on CRISPR/Cas9.

In some embodiments, the non-human mammal is mouse. In some embodiments,the mouse is a C57BL/6 mouse. In some embodiments, the non-human mammalin step (c) is a female with false pregnancy.

The disclosure also relates to a method for establishing agenetically-modified non-human animal expressing two human or chimeric(e.g., humanized) genes. The method includes the steps of

(a) using the method for establishing a PD-L1 gene humanized animalmodel to obtain a PD-L1 gene genetically modified humanized mouse;

(b) mating the PD-L1 gene genetically modified humanized mouse obtainedin step (a) with another humanized mouse, and then screening to obtain adouble humanized mouse model.

In some embodiments, in step (b), the PD-L1 gene genetically modifiedhumanized mouse obtained in step (a) is mated with a PD-1 humanizedmouse to obtain a PD-L1 and PD-1 double humanized mouse model.

The disclosure also relates to non-human mammal generated through themethods as described herein.

In some embodiments, the genome thereof contains human gene(s).

In some embodiments, the non-human mammal is a rodent. In someembodiments, the non-human mammal is a mouse.

In some embodiments, the non-human mammal expresses a protein encoded bya humanized PD-L1 gene.

The disclosure also relates to an offspring of the non-human mammal.

In another aspect, the disclosure relates to a tumor bearing non-humanmammal model, characterized in that the non-human mammal model isobtained through the method as described herein.

In some embodiments, the non-human mammal is a rodent. In someembodiments, the non-human mammal is a mouse.

The disclosure also relates to a cell or cell line, or a primary cellculture thereof derived from the non-human mammal or an offspringthereof, or the tumor bearing non-human mammal.

The disclosure further relates to the tissue, organ or a culture thereofderived from the non-human mammal or an offspring thereof, or the tumorbearing non-human mammal.

In another aspect, the disclosure relates to a tumor tissue derived fromthe non-human mammal or an offspring thereof when it bears a tumor, orthe tumor bearing non-human mammal.

In one aspect, the disclosure relates to a PD-L1 amino acid sequence ofa humanized mouse, wherein the amino acid sequence is selected from thegroup consisting of:

a) an amino acid sequence shown in SEQ ID NO: 33;

b) an amino acid sequence having a homology of at least 90% with theamino acid sequence shown in SEQ ID NO: 33;

c) an amino acid sequence encoded by a nucleic acid sequence, whereinthe nucleic acid sequence is able to hybridize to a nucleotide sequenceencoding the amino acid shown in SEQ ID NO: 33 under a low stringencycondition;

d) an amino acid sequence having a homology of at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or at least 99% with the amino acidsequence shown in SEQ ID NO: 33;

e) an amino acid sequence that is different from the amino acid sequenceshown in SEQ ID NO: 33 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or nomore than 1 amino acid; or

f) an amino acid sequence that comprises a substitution, a deletionand/or insertion of one or more amino acids to the amino acid sequenceshown in SEQ ID NO: 33.

The disclosure also relates to a PD-L1 DNA sequence of a humanizedmouse, wherein the DNA sequence is selected from the group consistingof:

a) a DNA sequence that encodes the PD-L1 amino acid sequence of ahumanized mouse;

b) a DNA sequence that is shown in SEQ ID NO: 32;

c) a DNA sequence having a CDS encoding sequence as shown in SEQ ID NO:31;

d) a DNA sequence that is able to hybridize to the nucleotide sequenceas shown in SEQ ID NO: 32 or SEQ ID NO: 31 under a low stringencycondition;

e) a DNA sequence that has a homology of at least 90% with thenucleotide sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 31;

f) a DNA sequence that encodes an amino acid sequence, wherein the aminoacid sequence has a homology of at least 90% with the amino acid shownin SEQ ID NO: 33;

g) a DNA sequence that encodes an amino acid sequence, wherein the aminoacid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or at least 99% with the amino acid sequence shown in SEQID NO: 33;

h) a DNA sequence that encodes an amino acid sequence, wherein the aminoacid sequence is different from the amino acid sequence shown in SEQ IDNO: 33 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1amino acid; and/or

i) a DNA sequence that encodes an amino acid sequence, wherein the aminoacid sequence comprises a substitution, a deletion and/or insertion of1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acids to the amino acidsequence shown in SEQ ID NO: 33.

The disclosure further relates to a PD-L1 genomic DNA sequence of ahumanized mouse, a DNA sequence obtained by a reverse transcription ofthe mRNA obtained by transcription thereof is consistent with orcomplementary to the DNA sequence; a construct expressing the amino acidsequence thereof; a cell comprising the construct thereof; a tissuecomprising the cell thereof.

The disclosure further relates to the use of the non-human mammal or anoffspring thereof, or the tumor bearing non-human mammal, the animalmodel generated through the method as described herein in thedevelopment of a product related to an immunization processes of humancells, the manufacture of a human antibody, or the model system for aresearch in pharmacology, immunology, microbiology and medicine.

The disclosure also relates to the use of the non-human mammal or anoffspring thereof, or the tumor bearing non-human mammal, the animalmodel generated through the method as described herein in the productionand utilization of an animal experimental disease model of animmunization processes involving human cells, the study on a pathogen,or the development of a new diagnostic strategy and/or a therapeuticstrategy.

The disclosure further relates to the use of the non-human mammal or anoffspring thereof, or the tumor bearing non-human mammal, the animalmodel generated through the methods as described herein, in thescreening, verifying, evaluating or studying the PD-L1 gene function,human PD-L1 antibodies, the drugs or efficacies for human PD-L1targeting sites, and the drugs for immune-related diseases and antitumordrugs.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the 5′ terminal target site sgRNA activitytest results (sgRNA1-sgRNA2, sgRNA4-sgRNA10), and the 3′ terminal targetsite sgRNA activity test results (sgRNA1l-sgRNA17). Con is a negativecontrol; and PC is a positive control.

FIG. 2 is a schematic diagram showing pT7-sgRNA plasmid map.

FIG. 3 is a schematic diagram showing comparison of human and mousePD-L1 genes.

FIG. 4 is a schematic diagram showing humanized PD-L1 mouse gene map.

FIG. 5 is a schematic diagram showing mouse PD-L1 gene targetingstrategy.

FIG. 6 shows pClon-4G-PD-L1 plasmid digestion result. M is the Marker,ck is the undigested plasmid control, no 1 and 2 indicate two clones ofpClo-4G-PD-L1.

FIG. 7A shows PCR identification result (5′ terminus) of samples frommouse tails (WT is wild type; M is marker, No. 4, 5, 7, 8 and 9 arepositive mice; and the unlabeled ones are negative mice).

FIG. 7B shows mouse tail PCR identification result (3′ terminus). (WT iswild type; + is positive control, No. 4, 5, 7, 8 and 9 are positivemice; and the unlabeled ones are negative mice).

FIG. 8 shows F1 generation mic Southern blot results (WT is wild type,No. 6, 7 mice have no random insertion).

FIGS. 9A-9E show flow cytometry analysis results for C57BL/6 mice (FIGS.9A, 9B, 9D) and PD-L1 humanized mice (FIGS. 9C, 9E). Anti-mouse CD3antibody was used to stimulate the T cell activation in the spleen, andthen anti-mouse (FIGS. 9B and 9C) and anti-human (FIGS. 9D and 9E) PD-L1antibodies with fluorescent labels were used for cell labeling. Comparedto the control group, the cells with the expression of human PD-L1protein can be detected in the spleen of PD-L1 humanized F1 hybrids;whereas in the spleen of C57BL/6 mice, no cells expressing human PD-L1protein were detected.

FIGS. 10A-10F show flow cytometry analysis results for two wild typeC57BL/6 mice and one B-hPD-L1 homozygous mouse, which were respectivelystimulated by anti-mouse CD3 antibody to stimulate T cell activation intheir spleens, and then anti-mouse PD-L1 antibody mPD-L1APC (FIGS. 10A,10B, 10C) and anti-human PD-L1 antibody hPD-L1PE (FIGS. 10D, 10E, 10F)were used for cell labeling, which were then detected in the flowcytometry analysis. Compared with the control group (FIGS. 10A, 10B,10D, 10E), the cells with the expression of human PD-L1 protein can bedetected in the spleens of B-hPD-L1 homozygous mouse (FIG. 10F); whereasin the spleen of C57BL/6 mouse, no cells expressing human PD-L1 proteinwere detected (FIGS. 10D, 10E).

FIG. 11 shows RT-PCR detection results, wherein +/+ is wild type C57BL/6mouse; H/H is B-hPD-L1 homozygous mouse; and GAPDH is an internalcontrol.

FIG. 12 shows identification results for gene knockout mice, wherein WTis wild type, the mice with no. EL15-76, EL15-77, EL15-78 areheterozygous mice.

FIG. 13. Mouse colon cancer cells MC38 were implanted into B-hPD-L1 miceand antitumor efficacy studies were performed using different doses ofhuman PD-L1 antibody Atezolizumab (1 mg/kg, 3 mg/kg and 10 mg/kg). Therewas no significant difference in mean weight gain between experimentalgroups G1 to G4.

FIG. 14. Mouse colon cancer cells MC38 were implanted into B-PD-L1-4mice and antitumor efficacy studies were performed using different dosesof human PD-L1 antibody atezolizumab (1 mg/kg, 3 mg/kg and 10 mg/kg).The average volume of tumor in the experimental group was significantlysmaller than that in G1 control group, and the differences weresignificant.

FIGS. 15A-15D show mouse tail PCR identification result, where + ispositive control, − is negative control (FIGS. 15A, 15B); WT is wildtype, −/− is humanized PD-1 homozygous mouse, +/− is humanized PD-1heterozygous mouse (FIGS. 15C, 15D). FIGS. 15A and 15B show that themice numbered 1-16 are homozygous for PD-L1 gene. FIGS. 16C and 16D showthat the mice numbered 1-16 are homozygous for PD-1 gene. The results ofthe two groups show that the 16 mice numbered 1-16 are homozygous forboth humanized PD-L1 and PD-1 genes.

FIGS. 16A-16F show flow cytometry analysis results, wherein C57BL/6 mice(FIGS. 16A, 16B, 16D, 16E) and double humanized PD-L1/PD-1 homozygotes(FIGS. 16C, 16F) were used. Anti-mouse CD3 antibody was used tostimulate T cell activation in the spleens of the mice, and then themouse PD-L1 antibody mPD-L1 APC (FIGS. 16A, 16B, 16C), human PD-L1antibody hPD-L1 PE (FIGS. 16D, 16E, 16F), and mouse T cell surfaceantibody mTcRβ were used to label T cell extracellular proteins. Theresults show that the cells expressing humanized PD-L1 proteins weredetected in the spleens of double humanized PD-L1/PD-1 homozygotes;while no cells expressing humanized PD-L1 were detected in the spleensof C57BL/6 control mice.

FIGS. 17A-17F show flow cytometry analysis results, wherein C57BL/6 mice(FIGS. 17A, 17B, 17D, 17E) and double humanized PD-L1/PD-1 homozygousmice (FIGS. 17C, 17F) were used. Anti-mouse CD3 antibody was used tostimulate T cell activation in the spleens of the mice, and then themouse PD-1 antibody mPD-1 PE (FIGS. 17A, 17B, 17C), human PD-1 antibodyhPD-1 FITC (FIGS. 17D, 17E, 17F), and mouse T cell surface antibodymTcRβ were used to label T cell extracellular proteins. The results showthat the cells expressing human PD-1 proteins were detected in thespleens of double humanized PD-L1/PD-1 homozygous mice; while no cellsexpressing humanized PD-1 protein were detected in the spleen of C57BL/6control mice.

FIG. 18 shows RT-PCR detection results for PD-L1 expression, wherein +/+is wild type C57BL/6 mouse; H/H is humanized PD-1/PD-L1 homozygousmouse; and GAPDH is an internal control.

FIG. 19 shows RT-PCR detection results for PD-1 expression, wherein +/+is wild type C57BL/6 mouse; H/H is humanized PD-1/PD-L1 homozygousmouse; and GAPDH is an internal control.

FIG. 20 is a schematic diagram of the targeting strategy for embryonicstem cells.

FIG. 21 shows the alignment between mouse PD-L1 amino acid sequence(NP_068693.1; SEQ ID NO: 27) and human PD-L1 amino acid sequence(NP_054862.1; SEQ ID NO: 29).

DETAILED DESCRIPTION

This disclosure relates to transgenic non-human animal with human orchimeric (e.g., humanized) Programmed death-ligand 1 (PD-L1 or PDL1),and methods of use thereof.

Programmed death-ligand 1 (PD-L1) also known as cluster ofdifferentiation 274 (CD274) or B7 homolog 1 (B7-H1) is a protein that inhumans is encoded by the CD274 gene. PD-L1 is a type 1 transmembraneprotein that has been speculated to play a major role in suppressing theimmune system during particular events such as pregnancy, tissueallografts, autoimmune disease and other disease states such ashepatitis.

PD-1 (programmed death-1) is mainly expressed on the surfaces of T cellsand primary B cells; two ligands of PD-1 (PD-L1 and PD-L2) are widelyexpressed in antigen-presenting cells (APCs). The interaction of PD-1with its ligands plays an important role in the negative regulation ofthe immune response. PD-L1 protein expression can be detected in manyhuman tumor tissues. The microenvironment of tumor site can induce theexpression of PD-L1 on tumor cells. PD-L1 expression is beneficial tothe occurrence and growth of tumors. It is able to induce apoptosis ofantitumor T cells, and thus allow tumor to escape from the immune systemattacks. Inhibition the binding between PD-1 and its ligand can make thetumor cells exposed to the killing effect of the immune system, and thuscan reach the effect of killing tumor tissues and treating cancers.

Experimental animal disease model is an indispensable research tool forstudying the etiology, pathogenesis of the disease, as well as thedevelopment of prevention and control techniques and therapeutic drugsfor the disease. Common experimental animals include mice, rats, guineapigs, hamsters, rabbits, dogs, monkeys, pigs, fish and so on. However,there are many differences between human and animal genes and proteinsequences, and many human proteins cannot bind to the animal'shomologous proteins to produce biological activity, leading to that theresults of many clinical trials do not match the results obtained fromanimal experiments. A large number of clinical studies are in urgentneed of better animal models. With the continuous development andmaturation of genetic engineering technologies, the use of human cellsor genes to replace or substitute an animal's endogenous similar cellsor genes to establish a biological system or disease model closer tohuman, and establish the humanized experimental animal models (humanizedanimal model) has provided an important tool for new clinical approachesor means. In this context, the genetically engineered animal model, thatis, the use of genetic manipulation techniques, the use of human normalor mutant genes to replace animal homologous genes, can be used toestablish the genetically modified animal models that are closer tohuman gene systems. The humanized animal models not only have variousimportant applications. Due to the presence of human or humanized genes,the animals can express or express in part of the proteins with humanfunctions, so as to greatly reduce the differences in clinical trialsbetween humans and animals, and provide the possibility of drugscreening at animal levels.

Unless otherwise specified, the practice of the methods described hereincan take the techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA andimmunology. These techniques are explained in detail in the followingliterature, for examples: Molecular Cloning A Laboratory Manual, 2ndEd., ed. By Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered.,1985); Oligonucleotide Synthesis (M. J. Gaited., 1984); Mullis et alU.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J.Higginseds. 1984); Transcription And Translation (B. D. Hames & S. J.Higginseds. 1984); Culture Of Animal Cell (R. I. Freshney, Alan R. Liss,Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,A Practical Guide To Molecular Cloning (1984), the series, Methods InENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press,Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) andVol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Caloseds., 1987,Cold Spring Harbor Laboratory); Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Hand book Of Experimental Immunology, Volumes V (D. M. Weir andC. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986), each ofwhich is incorporated herein in its entirety by reference.

Programmed Death-Ligand 1 (PD-L1)

Programmed death-ligand 1 (PD-L1) is a ligand of Programmed death 1(PD-1). The microenvironment of tumor site can induce the expression ofPD-1 on tumor cells, allowing tumor to escape from the immune systemattacks. Inhibiting the binding between PD-L1 and PD-1 exposes tumorcells to the immune system, and thus can kill tumor tissues and treatcancers.

In human genomes, PD-L1 gene locus has 7 exons, non-coding exon 1, exon2, exon 3, exon 4, exon 5, exon 6, and exon 7 (FIG. 3). The PD-L1protein also has an extracellular region, a transmembrane region, and acytoplasmic region. The nucleotide sequence for human PD-L1mRMA is NM014143.3 (SEQ ID NO: 28), the amino acid sequence for human PD-L1 isNP_054862.1 (SEQ ID NO: 29). The location for each exon and each regionin human PD-L1 nucleotide sequence and PD-L1 protein is listed below:

TABLE 1 Human PD-L1 NM_014143.3 NP_054862.1 (approximate location) (SEQID NO: 28) (SEQ ID NO: 29) Exon 1 1-94 (non-coding) Non-coding Exon 2 95-160  1-17 Exon 3 161-502  18-131 Exon 4 503-790 132-227 Exon 5791-898 228-263 Exon 6 899-958 264-283 Exon 7  959-3686 284-290 Signalpeptide 109-162  1-18 Extracellular region 163-825  19-239 (excludingsignal peptide region) Transmembrane region 826-888 240-260 Cytoplasmicregion 889-978 261-290 Donor region in Example 2 169-492  21-128 (Pointmutation at position 255 C to T)

Similarly, in mice, the PD-L1 gene locus has 7 exons as well, non-codingexon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and exon 7 (FIG. 3). Themouse PD-L1 protein also has an extracellular region, a transmembraneregion, and a cytoplasmic region, and the signal peptide is located atthe extracellular region of PD-L1. The nucleotide sequence for mousePD-L1mRNA is NM_021893.3 (SEQ ID NO: 26), the amino acid sequence formouse PD-L1 is NP_068693.1 (SEQ ID NO: 27). The location for each exonand each region in the mouse PD-L1 nucleotide sequence and amino acidsequence is listed below:

TABLE 2 Mouse PD-L1 NP_021893.3 NP_068693.1 (approximate location) (SEQID NO: 26) (SEQ ID NO: 27) Exon 1 1-69 (non-coding) Non-coding Exon 2 70-135  1-17 Exon 3 136-477  18-131 Exon 4 478-762 132-226 Exon 5763-873 227-263 Exon 6 874-933 264-283 Exon 7  934-3639 284-290 Signalpeptide  84-137  1-18 Extracellular region 138-800  19-239 (excludingsignal peptide region) Transmembrane region 801-863 240-260 Cytoplasmicregion 864-953 261-290 Replaced region in Example 2 144-467  21-128

The mouse PD-L1 gene (Gene ID: 60533) is located in Chromosome 19 of themouse genome, which is located from 29367438 to 29388095 of NC_000085.6(GRCm38.p4 (GCF_000001635.24)). The 5′-UTR is from 29367455 to 29367506and 29372454 to 29372467, the first intron is from 29367507 to 29372453,exon 2 is from 29372468 to 29372519, the second intron is from 29372520to 29373557, exon 3 is from 29373558 to 29373899, the third intron isfrom 29373900 to 29380303, exon 4 is from 29380304 to 29380588, thefourth intron is from 29380589 to 29382475, exon 5 is from 29382476 to29382586, the fifth intron is from 29382587 to 29384083, exon 6 is from29384084 to 29384143, the sixth intron is from 29384144 to 29385389,exon 7 is from 29385390 to 29385412, the 3′-UTR is from 29385413 to29388095, base on transcript NM_021893.3. All relevant information formouse PD-L1 locus can be found in the NCBI website with Gene ID: 60533,which is incorporated by reference herein in its entirety.

FIG. 21 shows the alignment between mouse PD-L1 amino acid sequence(NP_068693.1; SEQ ID NO: 27) and human PD-L1 amino acid sequence(NP_054862.1; SEQ ID NO: 29). Thus, the corresponding amino acid residueor region between human and mouse PD-L1 can also be found in FIG. 21.

PD-L1 genes, proteins, and locus of the other species are also known inthe art. For example, the gene ID for PD-L1 in Rattus norvegicus is499342, the gene ID for PD-L1 in Macaca mulatta (Rhesus monkey) is716043, the gene ID for PD-L1 in Sus scrofa (pig) is 574058. Therelevant information for these genes (e.g., intron sequences, exonsequences, amino acid residues of these proteins) can be found, e.g., inNCBI database.

The present disclosure provides human or chimeric (e.g., humanized)PD-L1 nucleotide sequence and/or amino acid sequences. In someembodiments, the entire sequence of mouse exon 1, exon 2, exon 3, exon4, exon 5, exon 6, exon 7, signal peptide, extracellular region,transmembrane region, and/or cytoplasmic region are replaced by thecorresponding human sequence. In some embodiments, a “region” or“portion” of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon7, signal peptide, extracellular region, transmembrane region, and/orcytoplasmic region are replaced by the corresponding human sequence. Theterm “region” or “portion” can refer to at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200,250, 300, 350, or 400 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 150 aminoacid residues. In some embodiments, the “region” or “portion” can be atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identicalto exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, signalpeptide, extracellular region, transmembrane region, or cytoplasmicregion. In some embodiments, a region, a portion, or the entire sequenceof mouse exon 2 and/or exon 3 is replaced by the human exon 2 and/orexon 3.

In some embodiments, the present disclosure also provides a chimeric(e.g., humanized) PD-L1 nucleotide sequence and/or amino acid sequences,wherein in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of thesequence are identical to or derived from mouse PD-L1 mRNA sequence(e.g., SEQ ID NO: 26), or mouse PD-L1 amino acid sequence (e.g., SEQ IDNO: 27); and in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of thesequence are identical to or derived from human PD-L1 mRNA sequence(e.g., SEQ ID NO: 28), or human PD-L1 amino acid sequence (e.g., SEQ IDNO: 29).

In some embodiments, the sequence encoding amino acids 21-128 of mousePD-L1 (SEQ ID NO: 27) is replaced. In some embodiments, the sequence isreplaced by a sequence encoding a corresponding region of human PD-L1(e.g., amino acids 21-128 of human PD-L1 (SEQ ID NO: 29)).

In some embodiments, the nucleic acids as described herein are operablylinked to a promotor or regulatory element, e.g., an endogenous mousePD-L1 promotor, an inducible promoter, an enhancer, and/or mouse orhuman regulatory elements.

In some embodiments, the nucleic acid sequence has at least a portion(e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous ornon-contiguous nucleotides) that are different from a portion of or theentire mouse PD-L1 nucleotide sequence (e.g., NM_021893.3 (SEQ ID NO:26)).

In some embodiments, the nucleic acid sequence has at least a portion(e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous ornon-contiguous nucleotides) that is the same as a portion of or theentire mouse PD-L1 nucleotide sequence (e.g., NM_021893.3 (SEQ ID NO:26)).

In some embodiments, the nucleic acid sequence has at least a portion(e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous ornon-contiguous nucleotides) that is different from a portion of or theentire human PD-L1 nucleotide sequence (e.g., NM_014143.3 (SEQ ID NO:28)).

In some embodiments, the nucleic acid sequence has at least a portion(e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous ornon-contiguous nucleotides) that is the same as a portion of or theentire human PD-L1 nucleotide sequence (e.g., NM_014143.3 (SEQ ID NO:28)).

In some embodiments, the amino acid sequence has at least a portion(e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguousor non-contiguous amino acid residues) that is different from a portionof or the entire mouse PD-L1 amino acid sequence (e.g., NP_068693.1 (SEQID NO: 27)).

In some embodiments, the amino acid sequence has at least a portion(e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguousor non-contiguous amino acid residues) that is the same as a portion ofor the entire mouse PD-L1 amino acid sequence (e.g., NP_068693.1 (SEQ IDNO: 27)).

In some embodiments, the amino acid sequence has at least a portion(e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguousor non-contiguous amino acid residues) that is different from a portionof or the entire human PD-L1 amino acid sequence (e.g., NP_054862.1 (SEQID NO: 29)).

In some embodiments, the amino acid sequence has at least a portion(e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguousor non-contiguous amino acid residues) that is the same as a portion ofor the entire human PD-L1 amino acid sequence (e.g., NP_054862.1 (SEQ IDNO: 29)).

The present disclosure also provides a humanized PD-L1 mouse amino acidsequence, wherein the amino acid sequence is selected from the groupconsisting of: a) an amino acid sequence shown in SEQ ID NO: 33;

b) an amino acid sequence having a homology of at least 90% with or atleast 90% identical to the amino acid sequence shown in SEQ ID NO: 33;

c) an amino acid sequence encoded by a nucleic acid sequence, whereinthe nucleic acid sequence is able to hybridize to a nucleotide sequenceencoding the amino acid shown in SEQ ID NO: 33 under a low stringencycondition;

d) an amino acid sequence having a homology of at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequenceshown in SEQ ID NO: 33;

e) an amino acid sequence that is different from the amino acid sequenceshown in SEQ ID NO: 33 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or nomore than 1 amino acid;

or

f) an amino acid sequence that comprises a substitution, a deletionand/or insertion of one or more amino acids to the amino acid sequenceshown in SEQ ID NO: 33.

The present disclosure also relates to a PD-L1 DNA sequence, wherein theDNA sequence can be selected from the group consisting of:

a) a DNA sequence as shown in SEQ ID NO: 31, or a DNA sequence encodinga homologous PD-L1 amino acid sequence of a humanized mouse;

b) a DNA sequence that is shown in SEQ ID NO: 32;

c) a DNA sequence that is able to hybridize to the nucleotide sequenceas shown in SEQ ID NO: 31 or SEQ ID NO: 32 under a low stringencycondition;

d) a DNA sequence that has a homology of at least 90% or at least 90%identical to the nucleotide sequence as shown in SEQ ID NO: 31 or SEQ IDNO: 32;

e) a DNA sequence that encodes an amino acid sequence, wherein the aminoacid sequence has a homology of at least 90% with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 33;

f) a DNA sequence that encodes an amino acid sequence, wherein the aminoacid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% with, or at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQID NO: 33;

g) a DNA sequence that encodes an amino acid sequence, wherein the aminoacid sequence is different from the amino acid sequence shown in SEQ IDNO: 33 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1amino acid; and/or

h) a DNA sequence that encodes an amino acid sequence, wherein the aminoacid sequence comprises a substitution, a deletion and/or insertion ofone or more amino acids to the amino acid sequence shown in SEQ ID NO:33.

The present disclosure further relates to a PD-L1 genomic DNA sequenceof a humanized mouse. The DNA sequence is obtained by a reversetranscription of the mRNA obtained by transcription thereof isconsistent with or complementary to the DNA sequence homologous to thesequence shown in SEQ ID NO: 32 or SEQ ID NO: 31.

The disclosure also provides an amino acid sequence that has a homologyof at least 90% with, or at least 90% identical to the sequence shown inSEQ ID NO: 33, and has protein activity. In some embodiments, thehomology with the sequence shown in SEQ ID NO: 33 is at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In someembodiments, the foregoing homology is at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, or at least about 59%.

In some embodiments, the percentage identity with the sequence shown inSEQ ID NO: 33 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least 99%. In some embodiments, the foregoing percentageidentity is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,or at least about 59%.

In some embodiments, the amino acid sequence (i) comprises an amino acidsequence; or (ii) consists of an amino acid sequence, wherein the aminoacid sequence comprises any one of the sequences mentioned above.

The disclosure also provides a nucleotide sequence that has a homologyof at least 90%, or at least 90% identical to the sequence shown in SEQID NO: 32, and encodes a polypeptide that has protein activity. In someembodiments, the homology with the sequence shown in SEQ ID NO: 32 is atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least99%. In some embodiments, the foregoing homology is at least about 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or at least about 59%.

In some embodiments, the percentage identity with the sequence shown inSEQ ID NO: 32 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least 99%. In some embodiments, the foregoing percentageidentity is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,or at least about 59%.

The disclosure also provides a nucleic acid sequence that is at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence asdescribed herein, and an amino acid sequence that is at least 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% identical to any amino acid sequence as described herein.In some embodiments, the disclosure relates to nucleotide sequencesencoding any peptides that are described herein, or any amino acidsequences that are encoded by any nucleotide sequences as describedherein. In some embodiments, the nucleic acid sequence is less than 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300,350, 400, or 500 nucleotides. In some embodiments, the amino acidsequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, or 150 amino acid residues.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The length of a reference sequence aligned for comparison purposes is atleast 80% of the length of the reference sequence, and in someembodiments is at least 90%, 95%, or 100%. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences. For purposes of the present disclosure, the comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a Blossum 62 scoring matrix with a gap penaltyof 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The term “percent homology” is often used to mean “sequence similarity.”The percentage of identical residues (percent identity) and thepercentage of residues conserved with similar physicochemical properties(percent similarity), e.g. leucine and isoleucine, are both used to“quantify the homology”. Residues conserved with similar physicochemicalproperties are well known in the art. The percent homology, in manycases, is higher than the percent identity.

Cells, tissues, and animals (e.g., mouse) are also provided thatcomprise the nucleotide sequences as described herein, as well as cells,tissues, and animals (e.g., mouse) that express human or humanized PD-L1from an endogenous non-human PD-L1 locus.

Genetically Modified Animals

As used herein, the term “genetically-modified non-human animal” refersto a non-human animal having exogenous DNA in at least one chromosome ofthe animal's genome. In some embodiments, at least one or more cells,e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50% of cells ofthe genetically-modified non-human animal have the exogenous DNA in itsgenome. The cell having exogenous DNA can be various kinds of cells,e.g., an endogenous cell, a somatic cell, an immune cell, a T cell, a Bcell, a germ cell, a blastocyst, or an endogenous tumor cell. In someembodiments, genetically-modified non-human animals are provided thatcomprise a modified endogenous PD-L1 locus that comprises an exogenoussequence (e.g., a human sequence), e.g., a replacement of one or morenon-human sequences with one or more human sequences. The animals aregenerally able to pass the modification to progeny, i.e., throughgermline transmission.

As used herein, the term “chimeric gene” or “chimeric nucleic acid”refers to a gene or a nucleic acid, wherein two or more portions of thegene or the nucleic acid are from different species, or at least one ofthe sequences of the gene or the nucleic acid does not correspond to thewildtype nucleic acid in the animal. In some embodiment, the chimericgene or chimeric nucleic acid has at least one portion of the sequencethat is derived from two or more different sources, e.g., sequencesencoding different proteins or sequences encoding the same (orhomologous) protein of two or more different species. In someembodiments, the chimeric gene or the chimeric nucleic acid is ahumanized gene or humanized nucleic acid.

As used herein, the term “chimeric protein” or “chimeric polypeptide”refers to a protein or a polypeptide, wherein two or more portions ofthe protein or the polypeptide are from different species, or at leastone of the sequences of the protein or the polypeptide does notcorrespond to wildtype amino acid sequence in the animal. In someembodiments, the chimeric protein or the chimeric polypeptide has atleast one portion of the sequence that is derived from two or moredifferent sources, e.g., same (or homologous) proteins of differentspecies. In some embodiments, the chimeric protein or the chimericpolypeptide is a humanized protein or a humanized polypeptide.

In some embodiments, the chimeric gene or the chimeric nucleic acid is ahumanized PD-L1 gene or a humanized PD-L1 nucleic acid. In someembodiments, at least one or more portions of the gene or the nucleicacid is from the human PD-L1 gene, at least one or more portions of thegene or the nucleic acid is from a non-human PD-L1 gene. In someembodiments, the gene or the nucleic acid comprises a sequence thatencodes a PD-L1 protein. The encoded PD-L1 protein is functional or hasat least one activity of the human PD-L1 protein or the non-human PD-L1protein, e.g., binding to human or non-human PD-1, regulate immuneresponse, and/or downregulate immune response when bound to PD-1.

In some embodiments, the chimeric protein or the chimeric polypeptide isa humanized PD-L1 protein or a humanized PD-L1 polypeptide. In someembodiments, at least one or more portions of the amino acid sequence ofthe protein or the polypeptide is from a human PD-L1 protein, and atleast one or more portions of the amino acid sequence of the protein orthe polypeptide is from a non-human PD-L1 protein. The humanized PD-L1protein or the humanized PD-L1 polypeptide is functional or has at leastone activity of the human PD-L1 protein or the non-human PD-L1 protein.

The genetically modified non-human animal can be various animals, e.g.,a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer,sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesusmonkey). For the non-human animals where suitable genetically modifiableES cells are not readily available, other methods are employed to make anon-human animal comprising the genetic modification. Such methodsinclude, e.g., modifying a non-ES cell genome (e.g., a fibroblast or aninduced pluripotent cell) and employing nuclear transfer to transfer themodified genome to a suitable cell, e.g., an oocyte, and gestating themodified cell (e.g., the modified oocyte) in a non-human animal undersuitable conditions to form an embryo. These methods are known in theart, and are described, e.g., in A. Nagy, et al., “Manipulating theMouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring HarborLaboratory Press, 2003, which is incorporated by reference herein in itsentirety.

In one aspect, the animal is a mammal, e.g., of the superfamilyDipodoidea or Muroidea. In some embodiments, the genetically modifiedanimal is a rodent. The rodent can be selected from a mouse, a rat, anda hamster. In some embodiment, the rodent is selected from thesuperfamily Muroidea. In some embodiments, the genetically modifiedanimal is from a family selected from Calomyscidae (e.g., mouse-likehamsters), Cricetidae (e.g., hamster, New World rats and mice, voles),Muridae (true mice and rats, gerbils, spiny mice, crested rats),Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy ratsand mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae(e.g., mole rates, bamboo rats, and zokors). In some embodiments, thegenetically modified rodent is selected from a true mouse or rat (familyMuridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment,the non-human animal is a mouse.

In some embodiments, the animal is a mouse of a C57BL strain selectedfrom C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J,C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, andC57BL/Ola. In some embodiments, the mouse is a 129 strain selected fromthe group consisting of a strain that is 129P1, 129P2, 129P3, 129X1,129S1 (e.g., 12951/SV, 12951/SvIm), 129S2, 129S4, 129S5, 12959/SvEvH,129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2. These mice aredescribed, e.g., in Festing et al., Revised nomenclature for strain 129mice, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment andChimera Analysis of 129/SvEv- and C57BL/6-Derived Mouse Embryonic StemCell Lines (2000), which is incorporated by reference in its entirety.In some embodiments, the genetically modified mouse is a mix of the 129strain and the C57BL/6 strain. In some embodiments, the mouse is a mixof the 129 strains, or a mix of the BL/6 strains. In some embodiment,the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments,the mouse is a mix of a BALB strain and another strain. In someembodiments, the mouse is from a hybrid line (e.g., 50% BALB/c-50%12954/Sv; or 50% C57BL/6-50% 129).

In some embodiments, the animal is a rat. The rat can be selected from aWistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain,F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mixof two or more strains selected from the group consisting of Wistar,LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

The animal can have one or more other genetic modifications, and/orother modifications, that are suitable for the particular purpose forwhich the humanized PD-L1 animal is made. For example, suitable mice formaintaining a xenograft (e.g., a human cancer or tumor), can have one ormore modifications that compromise, inactivate, or destroy the immunesystem of the non-human animal in whole or in part. Compromise,inactivation, or destruction of the immune system of the non-humananimal can include, for example, destruction of hematopoietic cellsand/or immune cells by chemical means (e.g., administering a toxin),physical means (e.g., irradiating the animal), and/or geneticmodification (e.g., knocking out one or more genes). Non-limitingexamples of such mice include, e.g., NOD mice, SCID mice, NON/SCID mice,IL2Rγ knockout mice, NOD/SCID/γcnull mice (Ito, M. et al.,NOD/SCID/γcnull mouse: an excellent recipient mouse model forengraftment of human cells, Blood 100(9):3175-3182, 2002), nude mice,and Rag1 and/or Rag2 knockout mice. These mice can optionally beirradiated, or otherwise treated to destroy one or more immune celltype. Thus, in various embodiments, a genetically modified mouse isprovided that can include a humanization of at least a portion of anendogenous non-human PD-L1 locus, and further comprises a modificationthat compromises, inactivates, or destroys the immune system (or one ormore cell types of the immune system) of the non-human animal in wholeor in part. In some embodiments, modification is, e.g., selected fromthe group consisting of a modification that results in NOD mice, SCIDmice, NOD/SCID mice, IL-2Rγ knockout mice, NOD/SCID/γc null mice, nudemice, Rag1 and/or Rag2 knockout mice, and a combination thereof. Thesegenetically modified animals are described, e.g., in US20150106961,which is incorporated by reference in its entirety. In some embodiments,the mouse can include a replacement of all or part of mature PD-L1coding sequence with human mature PD-L1 coding sequence.

Genetically modified non-human animals that comprise a modification ofan endogenous non-human PD-L1 locus. In some embodiments, themodification can comprise a human nucleic acid sequence encoding atleast a portion of a mature PD-L1 protein (e.g., at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical tothe mature PD-L1 protein sequence). Although genetically modified cellsare also provided that can comprise the modifications described herein(e.g., ES cells, somatic cells), in many embodiments, the geneticallymodified non-human animals comprise the modification of the endogenousPD-L1 locus in the germline of the animal.

Genetically modified animals can express a human PD-L1 and/or a chimeric(e.g., humanized) PD-L1 from endogenous mouse loci, wherein theendogenous mouse PD-L1 gene has been replaced with a human PD-L1 geneand/or a nucleotide sequence that encodes a region of human PD-L1sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%,50%, 60%, 70&, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thehuman PD-L1 sequence. In various embodiments, an endogenous non-humanPD-L1 locus is modified in whole or in part to comprise human nucleicacid sequence encoding at least one protein-coding sequence of a maturePD-L1 protein.

In some embodiments, the genetically modified mice express the humanPD-L1 and/or chimeric PD-L1 (e.g., humanized PD-L1) from endogenous locithat are under control of mouse promoters and/or mouse regulatoryelements. The replacement(s) at the endogenous mouse loci providenon-human animals that express human PD-L1 or chimeric PD-L1 (e.g.,humanized PD-L1) in appropriate cell types and in a manner that does notresult in the potential pathologies observed in some other transgenicmice known in the art. The human PD-L1 or the chimeric PD-L1 (e.g.,humanized PD-L1) expressed in animal can maintain one or more functionsof the wildtype mouse or human PD-L1 in the animal. For example, theexpressed PD-L1 can bind to human or non-human PD-1 and downregulateimmune response, e.g., downregulate immune response by at least 10%,20%, 30%, 40%, or 50%. Furthermore, in some embodiments, the animal doesnot express endogenous PD-L1. As used herein, the term “endogenousPD-L1” refers to PD-L1 protein that is expressed from an endogenousPD-L1 nucleotide sequence of a non-human animal (e.g., mouse) withoutthe genetic modification.

The genome of the animal can comprise a sequence encoding an amino acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human PD-L1 (NP_054862.1) (SEQ ID NO: 29). In someembodiments, the genome comprises a sequence encoding an amino acidsequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 33.

The genome of the genetically modified animal can comprise a replacementat an endogenous PD-L1 gene locus of a sequence encoding a region ofendogenous PD-L1 with a sequence encoding a corresponding region ofhuman PD-L1. In some embodiments, the sequence that is replaced is anysequence within the endogenous PD-L1 gene locus, e.g., exon 1, exon 2,exon 3, exon 4, exon 5, exon 6, exon 7, 5′-UTR, 3′UTR, the first intron,the second intron, the third intron, the fourth intron, the fifthintron, the sixth intron, etc. In some embodiments, the sequence that isreplaced is within the regulatory region of the endogenous PD-L1 gene.In some embodiments, the sequence that is replaced is exon 1, exon 2,exon 3, exon 4, exon 5, exon 6 and/or exon 7 of an endogenous mousePD-L1 gene locus.

The genetically modified animal can have one or more cells expressing ahuman or chimeric PD-L1 (e.g., humanized PD-L1) having an extracellularregion and a cytoplasmic region, wherein the extracellular regioncomprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region of human PD-L1. In someembodiments, the extracellular region of the humanized PD-L1 has asequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100amino acids (e.g., contiguously or non-contiguously) that are identicalto human PD-L1. Because human PD-L1 and non-human PD-L1 (e.g., mousePD-L1) sequences, in many cases, are different, antibodies that bind tohuman PD-L1 will not necessarily have the same binding affinity withmouse PD-L1 or have the same effects to mouse PD-L1. Therefore, thegenetically modified animal having a human or a humanized extracellularregion can be used to better evaluate the effects of anti-human PD-L1antibodies in an animal model. In some embodiments, the genome of thegenetically modified animal comprises a sequence encoding an amino acidsequence that corresponds to part or the entire sequence of exon 3 ofhuman PD-L1, part or the entire sequence of extracellular region ofhuman PD-L1 (with or without signal peptide), or part or the entiresequence of amino acids 21-128 of SEQ ID NO: 27.

In some embodiments, the non-human animal can have, at an endogenousPD-L1 gene locus, a nucleotide sequence encoding a chimerichuman/non-human PD-L1 polypeptide, wherein a human portion of thechimeric human/non-human PD-L1 polypeptide comprises a portion of humanPD-L1 extracellular domain, and wherein the animal expresses afunctional PD-L1 on a surface of a cell of the animal. The human portionof the chimeric human/non-human PD-L1 polypeptide can comprise a portionof exon 3 of human PD-L1. In some embodiments, the human portion of thechimeric human/non-human PD-L1 polypeptide can comprise a sequence thatis at least 80%, 85%, 90%, 95%, or 99% identical to amino acids 21-128of SEQ ID NO: 27.

In some embodiments, the non-human portion of the chimerichuman/non-human PD-L1 polypeptide comprises transmembrane and/orcytoplasmic regions of an endogenous non-human PD-L1 polypeptide. Insome embodiments, a few extracellular amino acids that are close to thetransmembrane region of PD-L1 are also derived from endogenous sequence.

Furthermore, the genetically modified animal can be heterozygous withrespect to the replacement at the endogenous PD-L1 locus, or homozygouswith respect to the replacement at the endogenous PD-L1 locus.

In some embodiments, the humanized PD-L1 locus lacks a human PD-L15′-UTR. In some embodiment, the humanized PD-L1 locus comprises a rodent(e.g., mouse) 5′-UTR. In some embodiments, the humanization comprises ahuman 3′-UTR. In appropriate cases, it may be reasonable to presume thatthe mouse and human PD-L1 genes appear to be similarly regulated basedon the similarity of their 5′-flanking sequence. As shown in the presentdisclosure, humanized PD-L1 mice that comprise a replacement at anendogenous mouse PD-L1 locus, which retain mouse regulatory elements butcomprise a humanization of PD-L1 encoding sequence, do not exhibitpathologies. Both genetically modified mice that are heterozygous orhomozygous for human PD-L1 are grossly normal.

The present disclosure further relates to a non-human mammal generatedthrough the method mentioned above. In some embodiments, the genomethereof contains human gene(s).

In some embodiments, the non-human mammal is a rodent, and preferably,the non-human mammal is a mouse.

In some embodiments, the non-human mammal expresses a protein encoded bya humanized PD-L1 gene.

In addition, the present disclosure also relates to a tumor bearingnon-human mammal model, characterized in that the non-human mammal modelis obtained through the methods as described herein. In someembodiments, the non-human mammal is a rodent (e.g., a mouse).

The present disclosure further relates to a cell or cell line, or aprimary cell culture thereof derived from the non-human mammal or anoffspring thereof, or the tumor bearing non-human mammal; the tissue,organ or a culture thereof derived from the non-human mammal or anoffspring thereof, or the tumor bearing non-human mammal; and the tumortissue derived from the non-human mammal or an offspring thereof when itbears a tumor, or the tumor bearing non-human mammal.

The present disclosure also provides non-human mammals produced by anyof the methods described herein. In some embodiments, a non-human mammalis provided; and the genetically modified animal contains the DNAencoding human PD-L1 in the genome of the animal.

In some embodiments, the non-human mammal comprises the geneticconstruct as shown in FIG. 2. In some embodiments, a non-human mammalexpressing human PD-L1 is provided. In some embodiments, thetissue-specific expression of human PD-L1 protein is provided.

In some embodiments, the expression of human PD-L1 in a geneticallymodified animal is controllable, as by the addition of a specificinducer or repressor substance.

Non-human mammals can be any non-human animal known in the art and whichcan be used in the methods as described herein. Preferred non-humanmammals are mammals, (e.g., rodents). In some embodiments, the non-humanmammal is a mouse.

Genetic, molecular and behavioral analyses for the non-human mammalsdescribed above can performed. The present disclosure also relates tothe progeny produced by the non-human mammal provided by the presentdisclosure mated with the same or other genotypes.

The present disclosure also provides a cell line or primary cell culturederived from the non-human mammal or a progeny thereof. A model based oncell culture can be prepared, for example, by the following methods.Cell cultures can be obtained by way of isolation from a non-humanmammal, alternatively cell can be obtained from the cell cultureestablished using the same constructs and the standard cell transfectiontechniques. The integration of genetic constructs containing DNAsequences encoding human PD-L1 protein can be detected by a variety ofmethods.

There are many analytical methods that can be used to detect exogenousDNA expression, including methods at the level of RNA (including themRNA quantification approaches using reverse transcriptase polymerasechain reaction (RT-PCR) or Southern blotting, and in situ hybridization)and methods at the protein level (including histochemistry, immunoblotanalysis and in vitro binding studies). In addition, the expressionlevel of the gene of interest can be quantified by ELISA techniques wellknown to those skilled in the art. Many standard analysis methods can beused to complete quantitative measurements. For example, transcriptionlevels can be measured using RT-PCR and hybridization methods includingRNase protection, Southern blot analysis, RNA dot analysis (RNAdot)analysis. Immunohistochemical staining, flow cytometry, Western blotanalysis can also be used to assess the presence of human PD-L1 protein.

Vectors

The present disclosure relates to a targeting vector, comprising: a) aDNA fragment homologous to the 5′ end of a region to be altered (5′arm), which is selected from the PD-L1 gene genomic DNAs in the lengthof 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding adonor region; and c) a second DNA fragment homologous to the 3′ end ofthe region to be altered (3′ arm), which is selected from the PD-L1 genegenomic DNAs in the length of 100 to 10,000 nucleotides.

In some embodiments, a) the DNA fragment homologous to the 5′ end of aconversion region to be altered (5′ arm) is selected from the nucleotidesequences that have at least 90% homology to the NCBI accession numberNC_000085.6; c) the DNA fragment homologous to the 3′ end of the regionto be altered (3′ arm) is selected from the nucleotide sequences thathave at least 90% homology to the NCBI accession number NC_000085.6.

In some embodiments, a) the DNA fragment homologous to the 5′ end of aregion to be altered (5′ arm) is selected from the nucleotides from theposition 29371941 to the position 29373565 of the NCBI accession numberNC_000085.6; c) the DNA fragment homologous to the 3′ end of the regionto be altered (3′ arm) is selected from the nucleotides from theposition 29373890 to the position 29375042 of the NCBI accession numberNC_000085.6.

In some embodiments, the length of the selected genomic nucleotidesequence in the targeting vector can be 1.5 kb and 1 kb.

In some embodiments, the region to be altered is exon 1, exon 2, exon 3,exon 4, exon 5, exon 6 and/or exon 7 of PD-L1 gene (e.g., exon 3 ofPD-L1 gene).

The targeting vector can further include a selected gene marker.

In some embodiments, the sequence of the 5′ arm is shown in SEQ ID NO:34; and the sequence of the 3′ arm is shown in SEQ ID NO: 42.

In some embodiments, the target region is derived from human. Forexample, the target region in the targeting vector is a part or entiretyof the nucleotide sequence of a human PD-L1, preferably the nucleotidesequence is shown as a first exon, a second exon, a third exon, or afourth exon or a fifth exon of the DNA sequence of the human PD-L1. Insome embodiments, the nucleotide sequence of the humanized PD-L1 encodesthe humanized PD-L1 protein with the NCBI accession number NP_054862.1.For example, the sequence of the target region can have the sequence asshown in SEQ ID NO: 37.

The disclosure also relates to a cell comprising the targeting vectorsas described above.

Moreover, the disclosure also relates to an sgRNA sequence forconstructing a humanized animal model, wherein the sgRNA sequencetargets the PD-L1 gene, the sgRNA is unique on the target sequence ofthe PD-L1 gene to be altered, and meets the sequence arrangement rule of5′-NNN (20) -NGG3′ or 5′-CCN-N(20)-3′; and in some embodiments, thetargeting site of the sgRNA in the mouse PD-L1 gene is located on theexon 1, exon 2, exon 3, exon 4, exon 5, exon 6 or exon 7 of the mousePD-L1 gene (e.g., exon 3 of the mouse PD-L1 gene).

In some embodiments, an upstream sequence thereof is shown as SEQ ID NO:17, and a downstream sequence thereof is shown as SEQ ID NO: 19, and thesgRNA sequence recognizes a 5′ targeting site. In some embodiments, theforward oligonucleotide sequence is obtained by adding TAGG to the 5′end of SEQ ID NO: 17; and the reverse oligonucleotide sequence isobtained by adding AAAC to the 5′ end of SEQ ID NO: 19.

In some embodiments, the disclosure provides an sgRNA sequence forconstructing a humanized animal model, wherein an upstream sequencethereof is shown as SEQ ID NO: 21, and a downstream sequence thereof isshown as SEQ ID NO: 23, and the sgRNA sequence recognizes a 3′ targetingsite. In some embodiments, the forward oligonucleotide sequence isobtained by adding TAGG to the 5′ end of SEQ ID NO: 21; and the reverseoligonucleotide sequence is obtained by adding AAAC to the 5′ end of SEQID NO: 23.

In some embodiments, the disclosure relates to a construct including thesgRNA sequence, and/or a cell including the construct.

In addition, the present disclosure further relates to a non-humanmammalian cell, having any one of the foregoing targeting vectors, andone or more in vitro transcripts of the sgRNA construct as describedherein. In some embodiments, the cell includes Cas9 mRNA or an in vitrotranscript thereof.

In some embodiments, the genes in the cell are heterozygous. In someembodiments, the genes in the cell are homozygous.

In some embodiments, the non-human mammalian cell is a mouse cell. Insome embodiments, the cell is a fertilized egg cell.

Methods of Making Genetically Modified Animals

Genetically modified animals can be made by several techniques that areknown in the art, including, e.g., nonhomologous end-joining (NHEJ),homologous recombination (HR), zinc finger nucleases (ZFNs),transcription activator-like effector-based nucleases (TALEN), and theclustered regularly interspaced short palindromic repeats (CRISPR)-Cassystem. In some embodiments, homologous recombination is used. In someembodiments, CRISPR-Cas9 genome editing is used to generate geneticallymodified animals. Many of these genome editing techniques are known inthe art, and is described, e.g., in Yin, Hao, Kevin J. Kauffman, andDaniel G. Anderson. “Delivery technologies for genome editing.” NatureReviews Drug Discovery 16.6 (2017): 387-399, which is incorporated byreference in its entirety. Many other methods are also provided and canbe used in genome editing, e.g., micro-injecting a genetically modifiednucleus into an enucleated oocyte, and fusing an enucleated oocyte withanother genetically modified cell.

Thus, in some embodiments, the disclosure provides replacing in at leastone cell of the animal, at an endogenous PD-L1 gene locus, a sequenceencoding a region of an endogenous PD-L1 with a sequence encoding acorresponding region of human or chimeric PD-L1. In some embodiments,the replacement occurs in a germ cell, a somatic cell, a blastocyst, ora fibroblast, etc. The nucleus of a somatic cell or the fibroblast canbe inserted into an enucleated oocyte.

FIG. 5 shows a humanization strategy for a mouse PD-L1 locus. In FIG. 5,the targeting strategy involves a vector comprising the 5′ endhomologous arm, human PD-L1 gene fragment, 3′ homologous arm. Theprocess can involve replacing endogenous PD-L1 sequence with humansequence by homologous recombination. In some embodiments, the cleavageat the upstream and the downstream of the target site (e.g., by zincfinger nucleases, TALEN or CRISPR) can result in DNA double strandsbreak, and the homologous recombination is used to replace endogenousPD-L1 sequence with human PD-L1 sequence.

Thus, in some embodiments, the methods for making a geneticallymodified, humanized animal, can include the step of replacing at anendogenous PD-L1 locus (or site), a nucleic acid encoding a sequenceencoding a region of endogenous PD-L1 with a sequence encoding acorresponding region of human PD-L1. The sequence can include a region(e.g., a part or the entire region) of exon 1, exon 2, exon 3, exon 4,exon 5, exon 6, and/or exon 7 of a human PD-L1 gene. In someembodiments, the sequence includes a region of exon 3 of a human PD-L1gene (e.g., amino acids 21-128 of SEQ ID NO: 29). In some embodiments,the region is located within the extracellular region of PD-L1. In someembodiments, the endogenous PD-L1 locus is exon 3 of mouse PD-L1.

In some embodiments, the methods of modifying a PD-L1 locus of a mouseto express a chimeric human/mouse PD-L1 peptide can include the steps ofreplacing at the endogenous mouse PD-L1 locus a nucleotide sequenceencoding a mouse PD-L1 with a nucleotide sequence encoding a humanPD-L1, thereby generating a sequence encoding a chimeric human/mousePD-L1.

In some embodiments, the nucleotide sequence encoding the chimerichuman/mouse PD-L1 can include a first nucleotide sequence encoding anextracellular region of mouse PD-L1 (with or without the mouse signalpeptide sequence); a second nucleotide sequence encoding anextracellular region of human PD-L1; a third nucleotide sequenceencoding a transmembrane and a cytoplasmic region of a mouse PD-L1.

In some embodiments, the nucleotide sequences as described herein do notoverlap with each other (e.g., the first nucleic tide sequence, thesecond nucleotide sequence, and/or the third nucleotide sequence do notoverlap). In some embodiments, the amino acid sequences as describedherein do not overlap with each other.

The present disclosure further provides a method for establishing aPD-L1 gene humanized animal model, comprising the following steps:

(a) providing the cell (e.g. a fertilized egg cell) based on the methodsdescribed herein;

(b) culturing the cell in a liquid culture medium;

(c) transplanting the cultured cell to the fallopian tube or uterus ofthe recipient female non-human mammal, allowing the cell to develop inthe uterus of the female non-human mammal;

(d) identifying the germline transmission in the offspring geneticallymodified humanized non-human mammal of the pregnant female in step (c).

In some embodiments, the non-human mammal in the foregoing method is amouse (e.g., a C57BL/6 mouse).

In some embodiments, the non-human mammal in step (c) is a female withpseudopregnancy (or false pregnancy).

In some embodiments, the fertilized eggs for the methods described aboveare C57BL/6 fertilized eggs. Other fertilized eggs that can also be usedin the methods as described herein include, but are not limited to,FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs andDBA/2 fertilized eggs.

Fertilized eggs can come from any non-human animal, e.g., any non-humananimal as described herein. In some embodiments, the fertilized eggcells are derived from rodents. The genetic construct can be introducedinto a fertilized egg by microinjection of DNA. For example, by way ofculturing a fertilized egg after microinjection, a cultured fertilizedegg can be transferred to a false pregnant non-human animal, which thengives birth of a non-human mammal, so as to generate the non-humanmammal mentioned in the method described above.

Methods of Using Genetically Modified Animals

Replacement of non-human genes in a non-human animal with homologous ororthologous human genes or human sequences, at the endogenous non-humanlocus and under control of endogenous promoters and/or regulatoryelements, can result in a non-human animal with qualities andcharacteristics that may be substantially different from a typicalknockout-plus-transgene animal. In the typical knockout-plus-transgeneanimal, an endogenous locus is removed or damaged and a fully humantransgene is inserted into the animal's genome and presumably integratesat random into the genome. Typically, the location of the integratedtransgene is unknown; expression of the human protein is measured bytranscription of the human gene and/or protein assay and/or functionalassay. Inclusion in the human transgene of upstream and/or downstreamhuman sequences are apparently presumed to be sufficient to providesuitable support for expression and/or regulation of the transgene.

In some cases, the transgene with human regulatory elements expresses ina manner that is unphysiological or otherwise unsatisfactory, and can beactually detrimental to the animal. The disclosure demonstrates that areplacement with human sequence at an endogenous locus under control ofendogenous regulatory elements provides a physiologically appropriateexpression pattern and level that results in a useful humanized animalwhose physiology with respect to the replaced gene are meaningful andappropriate and context of the humanized animal's physiology.

Genetically modified animals that express human or humanized PD-L1protein, e.g., in a physiologically appropriate manner, provide avariety of uses that include, but are not limited to, developingtherapeutics for human diseases and disorders, and assessing theefficacy of these human therapeutics in the animal models.

In various aspects, genetically modified animals are provided thatexpress human or humanized PD-L1, which are useful for testing agentsthat can decrease or block the interaction between PD-L1 and PD-1,testing whether an agent can increase or decrease the immune response,and/or determining whether an agent is an PD-1 or PD-L1 agonist orantagonist. The genetically modified animals can be, e.g., an animalmodel of a human disease, e.g., the disease is induced genetically (aknock-in or knockout). In various embodiments, the genetically modifiednon-human animals further comprise an impaired immune system, e.g., anon-human animal genetically modified to sustain or maintain a humanxenograft, e.g., a human solid tumor or a blood cell tumor (e.g., alymphocyte tumor, e.g., a B or T cell tumor).

In some embodiments, the genetically modified animals can be used fordetermining effectiveness of an anti-PD-L1 antibody for the treatment ofcancer. The methods involving administering the anti-PD-L1 antibody tothe animal as described herein, wherein the animal has a tumor; anddetermining the inhibitory effects of the anti-PD-L1 antibody to thetumor. The inhibitor effects that can be determined include, e.g., adecrease of tumor size or tumor volume, a decrease of tumor growth, areduction of the increase rate of tumor volume in a subject (e.g., ascompared to the rate of increase in tumor volume in the same subjectprior to treatment or in another subject without such treatment), adecrease in the risk of developing a metastasis or the risk ofdeveloping one or more additional metastasis, an increase of survivalrate, and an increase of life expectancy, etc. The tumor volume in asubject can be determined by various methods, e.g., as determined bydirect measurement, Mill or CT.

In some embodiments, the aminal comprises one or more cells (e.g.,immune cells, T cells) that express PD-1. In some embodiments, the tumorcomprises one or more cancer cells (e.g., human or mouse cancer cells)that are injected into the animal.

In some embodiments, the genetically modified animals can be used fordetermining whether an anti-PD-L1 antibody is an PD-1 or PD-L1 agonistor antagonist. In some embodiments, the methods as described herein arealso designed to determine the effects of the agent (e.g., anti-PD-L1antibodies) on PD-L1, e.g., whether the agent can stimulate T cells orinhibit T cells, whether the agent can upregulate the immune response ordownregulate immune response. In some embodiments, the geneticallymodified animals can be used for determining the effective dosage of atherapeutic agent for treating a disease in the subject, e.g., cancer.

The inhibitory effects on tumors can also be determined by methods knownin the art, e.g., measuring the tumor volume in the animal, and/ordetermining tumor (volume) inhibition rate (TGI_(TV)). The tumor growthinhibition rate can be calculated using the formula TGI_(TV)(%)=(1−TVt/TVc)×100, where TVt and TVc are the mean tumor volume (orweight) of treated and control groups.

In some embodiments, the anti-PD-L1 antibody is designed for treatingvarious cancers. As used herein, the term “cancer” refers to cellshaving the capacity for autonomous growth, i.e., an abnormal state orcondition characterized by rapidly proliferating cell growth. The termis meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. The term “tumor” as used herein refers to cancerous cells,e.g., a mass of cancerous cells. Cancers that can be treated ordiagnosed using the methods described herein include malignancies of thevarious organ systems, such as affecting lung, breast, thyroid,lymphoid, gastrointestinal, and genito-urinary tract, as well asadenocarcinomas which include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus. In some embodiments, the agents describedherein are designed for treating or diagnosing a carcinoma in a subject.The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. In some embodiments, thecancer is renal carcinoma or melanoma. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures. The term “sarcoma” is art recognized and refers to malignanttumors of mesenchymal derivation.

In some embodiments, the anti-PD-L1 antibody is designed for thetreating melanoma, non-small cell lung carcinoma (NSCLC), small celllung cancer (SCLC), bladder cancer, and/or prostate cancer (e.g.,metastatic hormone-refractory prostate cancer). Anit-PD-L1 antibodiesare known in the art, and include, e.g., atezolizumb, andare describedin, e.g., WO/2016/111645A1, WO/2016/022630, and US 20150322153, each ofwhich is incorporated by reference in its entirety.

The present disclosure also relates to the use of the animal modelgenerated through the method as described herein in the development of aproduct related to an immunization processes of human cells, themanufacturing of a human antibody, or the model system for a research inpharmacology, immunology, microbiology and medicine.

In some embodiments, the disclosure provides the use of the animal modelgenerated through the method as described herein in the production andutilization of an animal experimental disease model of an immunizationprocesses involving human cells, the study on a pathogen, or thedevelopment of a new diagnostic strategy and/or a therapeutic strategy.

The disclosure also relates to the use of the animal model generatedthrough the method mentioned above in the screening, verifying,evaluating or studying the PD-L1 gene function, human PD-L1 antibodies,drugs for human PD-L1 targeting sites, the drugs or efficacies for humanPD-L1 targeting sites, the drugs for immune-related diseases andantitumor drugs.

Genetically Modified Animal Model with Two or More Human or ChimericGenes

The present disclosure further relates to methods for geneticallymodified animal model with two or more human or chimeric genes. Theanimal can comprise a human or chimeric PD-L1 gene and a sequenceencoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein can beprogrammed cell death protein 1 (PD-1), TNF Receptor Superfamily Member4 (OX40), Lymphocyte Activating 3 (LAG-3), T-Cell Immunoglobulin AndMucin Domain-Containing Protein 3 (TIM-3), cytotoxicT-lymphocyte-associated protein 4 (CTLA-4), TNF Receptor SuperfamilyMember 9 (4-1BB), CD27, CD28, CD47, T-Cell Immunoreceptor With Ig AndITIM Domains (TIGIT), CD27, Glucocorticoid-Induced TNFR-Related Protein(GITR), or B And T Lymphocyte Associated (BTLA).

The methods of generating genetically modified animal model with two ormore human or chimeric genes (e.g., humanized genes) can include thefollowing steps:

(a) using the methods of introducing human PD-L1 gene or chimeric PD-L1gene as described herein to obtain a genetically modified non-humananimal;

(b) mating the genetically modified non-human animal with anothergenetically modified non-human animal, and then screening the progeny toobtain a genetically modified non-human animal with two or more human orchimeric genes.

In some embodiments, in step (b) of the method the genetically modifiedanimal can be mated with a genetically modified non-human animal withhuman or chimeric PD-1, OX40, LAG-3, TIM-3, CTLA-4, 4-1BB, CD27, CD28,CD47, TIGIT, CD27, GITR, or BTLA.

In some embodiments, the PD-L1 humanization is directly performed on agenetically modified animal having a human or chimeric PD-1, OX40,LAG-3, TIM-3, CTLA-4, 4-1BB, CD27, CD28, CD47, TIGIT, CD27, GITR, orBTLA gene.

As these proteins may involve different mechanisms, a combinationtherapy that targets two or more of these proteins thereof may be a moreeffective treatment. In fact, many related clinical trials are inprogress and have shown a good effect. The genetically modified animalmodel with two or more human or humanized genes can be used fordetermining effectiveness of a combination therapy that targets two ormore of these proteins, e.g., an anti-PD-L1 antibody and an additionaltherapeutic agent for the treatment of cancer. The methods includeadministering the anti-PD-L1 antibody and the additional therapeuticagent to the animal, wherein the animal has a tumor; and determining theinhibitory effects of the combined treatment to the tumor.

In some embodiments, the animal further comprises a sequence encoding ahuman or humanized programmed cell death protein 1 (PD-1). In someembodiments, the additional therapeutic agent is an anti-PD-1 antibody(e.g., nivolumab, pembrolizumab, avelumab, durvalumab, atezolizumab). Insome embodiments, the tumor comprises one or more tumor cells thatexpress CD80, CD86, PD-L1 or PD-L2.

In some embodiments, the combination treatment is designed for treatingvarious cancer as described herein, e.g., melanoma, non-small cell lungcarcinoma (NSCLC), small cell lung cancer (SCLC), bladder cancer,prostate cancer (e.g., metastatic hormone-refractory prostate cancer),breast cancer, and/or locally advanced or metastatic urothelialcarcinoma (e.g., whose conditions were still in progress in the 12-monthperiod of, before or after the treatment of platinum-based chemotherapyor receiving a new or adjuvant platinum chemotherapy).

In some embodiments, the methods described herein can be used toevaluate the combination treatment with some other methods. The methodsof treating a cancer that can be used alone or in combination withmethods described herein, include, e.g., treating the subject withchemotherapy, e.g., campothecin, doxorubicin, cisplatin, carboplatin,procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide,melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin,daunorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil,podophyllotoxin, tamoxifen, taxol, transplatinum, 5-flurouracil,vincristin, vinblastin, and/or methotrexate. Alternatively or inaddition, the methods can include performing surgery on the subject toremove at least a portion of the cancer, e.g., to remove a portion of orall of a tumor(s), from the patient.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Materials and Methods

The following materials were used in the following examples.

Ambion™ in vitro transcription kit was purchased from Ambion. Catalognumber is AM1354.

E. coli TOP10 competent cells were purchased from the Tiangen Biotech(Beijing) Co. Catalog number is CB104-02.

EcoRI, BamHI, NcoI, BglII, NdeI, HindIII, PstI were purchased from NEB.Catalog numbers are R3101M, R3469M, R3193M, R0144M, R0111V, R3104M, andR3140M.

Kanamycin was purchased from Amresco. Catalog number is 0408.

Cas9 mRNA was obtained from SIGMA. Catalog number is CAS9MRNA-1EA.

AIO kit was obtained from Beijing Biocytogen Co., Ltd. Catalog number isBCG-DX-004.

UCA kit was obtained from Beijing Biocytogen Co., Ltd. Catalog number isBCG-DX-001.

C57BL/6 mice were purchased from the China Food and Drugs ResearchInstitute National Rodent Experimental Animal Center.

Reverse Transcription Kit was obtained from TakaRa. Catalog number is6110A.

Mouse colon cancer cell line MC38 was purchased from Shanghai EnzymeResearch Biotechnology Co., Ltd.

Mouse CD3 antibody was obtained from BD. Catalog number is 563123.

mTcRβ PerCP was obtained from Biolegend. Catalog number is 109228.

mPD-1_PE was obtained from Biolegend. Catalog number is 109104.

hPD-1 FITC was obtained from Biolegend. Catalog number is 329904.

mPD-L1 APC was obtained from Biolegend. Catalog number is 124312.

hPD-L1 PE was obtained from Biolegend. Catalog number is 29706.

Example 1: Construction of pT7-sgRNA-PD-L1 and pT7-sgRNA-PD-L11

The target sequence determines the targeting specificity of small guideRNA (sgRNA) and the efficiency of Cas9 cleavage at the target gene.Therefore, target sequence selection is important for sgRNA vectorconstruction.

The 5′-terminal targeting sites (sgRNA1 to sgRNA8) and the 3′-terminaltargeting sites (sgRNA9 to sgRNA17) were designed and synthesized. The5′-terminal targeting sites and the 3′-terminal targeting sites are bothlocated on exon 3 of mouse PD-L1 gene, and the targeting site sequenceon PD-L1 of each sgRNA is as follows:

sgRNA-1 targeting sequence (SEQ ID NO: 1): 5′-gtatggcagcaacgtcacgatgg-3′sgRNA-2 targeting sequence (SEQ ID NO: 2): 5′-gcttgcgttagtggtgtactggg-3′sgRNA-4 targeting sequence (SEQ ID NO: 3): 5′-gctggacctgcttgcgttagtgg-3′sgRNA-5 targeting sequence (SEQ ID NO: 4): 5′-aggtccagctcccgttctacagg-3′sgRNA-6 targeting sequence (SEQ ID NO: 5): 5′-gtttactatcacggctccaaagg-3′sgRNA-7 targeting sequence (SEQ ID NO: 6): 5′-ggctccaaaggacttgtacgtgg-3′sgRNA-8 targeting sequence (SEQ ID NO: 7): 5′-cgtgatagtaaacgctgaaaagg-3′sgRNA-9 targeting sequence (SEQ ID NO: 8): 5′-attccctgtagaacgggagctgg-3′sgRNA-10 targeting sequence (SEQ ID NO: 9):5′-gacttgtacgtggtggagtatgg-3′sgRNA-11 targeting sequence (SEQ ID NO: 10):5′-tgctgcataatcagctacggtgg-3′sgRNA-12 targeting sequence (SEQ ID NO: 11):5′-cataatcagctacggtggtgcgg-3′sgRNA-13 targeting sequence (SEQ ID NO: 12):5′-gacgtcaagctgcaggacgcagg-3′sgRNA-14 targeting sequence (SEQ ID NO: 13):5′-tactgctgcataatcagctacgg-3′sgRNA-15 targeting sequence (SEQ ID NO: 14):5′-gatcacagacgtcaagctgcagg-3′sgRNA-16 targeting sequence (SEQ ID NO: 15):5′-gcttgacgtctgtgatctgaagg-3′sgRNA-17 targeting sequence (SEQ ID NO: 16):5′-cagcatttcccttcaaaagctgg-3′

The UCA kit was used to detect the activities of sgRNAs (FIG. 1). Theresults show that the guide sgRNAs have different activities. Two ofthem (sgRNA1 and sgRNA11, respectively) were selected for follow-upexperiments. TAGG was added to the 5′ end to obtain a forwardoligonucleotide sequence, and its complementary strand was added withAAAC to obtain a reverse oligonucleotide sequence. After annealing, theywere respectively digested by restriction enzyme (BbsI) and ligated topT7-sgRNA plasmid to obtain the expression vectors pT7-sgRNA-PDL1 andpT7-sgRNA-PDL11.

TABLE 3 sgRNA1 and sgRNA11 sequences sgRNA1 sequences SEQ ID NO: 17Upstream: 5′-TATGGCAGCAACGT CACGA-3′ SEQ ID NO: 18 (adding TAGGUpstream: to obtain a forward 5′-TAGGTAGGTATGGColigonucleotide sequence) AGCAACGTCACGA-3′ SEQ ID NO: 19 Downstream:5′-TCGTGACGTTGCT GCCATA-3′ SEQ ID NO: 20 Downstream:(complementary strand was 5′-AAACTCGTGACGT added with AAAC to obtainTGCTGCCATA-3′ a reverse oligonucleotide sequence) sgRNA11 SEQ ID NO: 21Upstream: 5′-CTGCATAATCAGC TACGG-3′ SEQ ID NO: 22 (adding TAGG Upstream:to obtain a forward 5′-TAGGCTGCATAAT oligonucleotide sequence)CAGCTACGG-3′ SEQ ID NO: 23 Downstream: 5′-CCGTAGCTGATTA TGCAG-3′SEQ ID NO: 24 Downstream: (complementary strand was 5′-AAACCCGTAGCTGadded with AAAC to obtain ATTATGCAG-3′ a reverse oligonucleotidesequence)

The Ligation Reaction:

TABLE 4 The ligation reaction conditions Double stranded fragment 1 μL(0.5 μM) pT7-sgRNA vector 1 μL (10 ng) T4 DNA Ligase 1 μL (5 U) 10 × T4DNA Ligase buffer 1 μL 50% PEG4000 1 μL H₂O Add to 10 μL

Reaction Conditions:

The ligation reaction was carried out at room temperature for 10 to 30min. The ligation product was then transferred to 30 μL of TOP10competent cells. The cells were then plated on a petri dish withKanamycin, and then cultured at 37° C. for at least 12 hours and thentwo clones were selected and added to LB medium with Kanamycin (5 ml),and then cultured at 37° C. at 250 rpm for at least 12 hours.

Randomly selected clones were sequenced, so as to verify theirsequences. The correct expression vectors pT7-sgRNA-PDL1 andpT7-sgRNA-PDL11 were selected for subsequent experiments.

Source of pT7-sgRNA Plasmid

PT7-sgRNA vector map is shown in FIG. 2. The plasmid backbone wasobtained from Takara (Catalog No. 3299). The DNA fragment containing T7promoter and sgRNA scaffold (SEQ ID NO: 25) was synthesized by a plasmidsynthesis company, and linked to the backbone vector by restrictionenzyme digestion (EcoRI and BamHI) and ligation. The target plasmid wasconfirmed by the sequencing results.

The DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ IDNO: 25):

gaattctaatacgactcactatagggggtcttcgagaagacctgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttaaaggatcc

Example 2. Construction of Vector pClon-4G-PDL1

A partial coding sequence of the mouse PD-L1 gene (Gene ID: 60533) fromexon 3 (based on the transcript of NCBI accession numberNM_021893.3→NP_068693.1 whose mRNA sequence is shown in SEQ ID NO: 26,and the corresponding protein sequence is shown in SEQ ID NO: 27) wasreplaced with a corresponding coding sequence of human homologous PD-L1gene (Gene ID: 29126) (based on the transcript of NCBI accession numberNM_014143.3→NP_054862.1, whose mRNA sequence was shown in SEQ ID NO: 28,and the corresponding protein sequence is shown in SEQ ID NO: 29). Thecomparison between the mouse PD-L1 and human PD-L1 is shown in FIG. 3,and the finally obtained humanized PD-L1 gene is shown in FIG. 4. Thehumanized mouse PD-L1 gene DNA sequence (chimeric PD-L1 gene DNA) isshown in SEQ ID NO: 30 (shown immediately below):

gcgtttact gtcacggttcccaaggacctatatgtggtagagtatggtagcaatatgacaattgaatgcaaattcccagtagaaaaacaattagaTctggctgcactaattgtctattgggaaatggaggataagaacattattcaatttgtgcatggagaggaagacctgaaggttcagcatagtagctacagacagagggcccggctgttgaaggaccagctctccctgggaaatgctgcacttcagatcacagatgtgaaattgcaggatgcaggggtgtaccgctgcatgatcagctatggtggtgccgactacaagcgaattactgtg aaa gtcaatgg

SEQ ID NO: 30 lists only the portion of DNA sequence involved in themodification, wherein the italicized underlined region is the humanPD-L1 gene sequence fragment.

The coding region sequence, mRNA sequence and the encoded proteinsequence thereof of the humanizedPD-L1 are respectively shown in SEQ IDNO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.

A targeting strategy involving a vector comprising the 5′ end homologousarm, human PD-L1 gene fragment, 3′ homologous arm as shown in FIG. 5 isalso developed. The process is as follows:

(1). Design upstream primers of homologous recombination fragments, anddownstream primers matching therewith, as well as other relatedsequences. Specifically:

5′ end homologous arm (SEQ ID NO: 34), nucleotide sequence of thepositions from 29371941 to 29373565 of the NCBI accession numberNC_000085.6 as follows:

atcatgtggaataggtgcgaggcagaggtgagattttaatggggaggaagcattgaaaagtgaaagtgaaaattggatgctctttgctttgaagctttgcctaaagcaggttttagctttcaaatacgtttcaatgttgaaagaacgcatgatacatatggaggggggcctggggggggtccttggctgagtttgaatgtacattaacaatctggggggctaataactcaatgtaaagctgctgatcccatcatactgacttctttccacttggttctacatggctttgagttacaaaatgaaagcattgaattttgaactgttcagctgtgtttccacacttgcaaatcggttgttggccagccctcagaattgcttcagttacagctggctcgtctgctctttccagactggcttttagggcttatgtatatatgagaaggacacatttactagtgtctccttgctctgctattgaaattaagcagacctctctgtgtttcccgttactagatagttcccaaaacatgaggatatttgctggcattatattcacagcctgctgtcacttgctacggggtaagtcaccaaatcttttcagtgggttctatattttcaatattttagctatgaattaaaaatggaagtaatttgtggggtgtgtatgtgtgtgtatatgtgtgtgtagaggggggtctgtgtgtatgtgcagttgctaggcacacataaagcgttcataggacaacctagagcttagtcctcaccttctaccttgtttgagacaaggtctcttatttgttgtacattgctgagtcctgtagttcggctagctcagaacctcctgggggctctcctgtctccacctcccagtccactgagattgtaggcacatgctactgcacctggcttctacctggtctctggggatttgaacttgggtccatgggctacacagcaagtcgtttacttactgggcaatcactccatcccctaagataattataaggaatataccttgcttatccaaacacattctcattctcctttgccataaataagttacttggcaaatatattgtatgtatttttaataaataaataaaatcttaaaaataaataaaattatttgtgaagacaaaaaaaataagttacttggaaaggatgaaggaaaatactggagattgggtgtggtttagtagtagaacacttggctgatgtaaaaaaaaagccctaggtgcaatcccaacaccagaaacaaatgaaggaatgaacaacaaccgcccccaccccccaggggatgaatataaaaatatcaggtaatacagaactaacaggtgatccgtttcctatgaataactactgaacattcccagggaggtggcccactgataatatatttttatttattggttccttttaaacaagactgggaatatattatctagcttgcatcaccaccaccaccccccacccccgccccatgaagttatttcaaagaagaattttagtgttcatgtgattccctaaataaaatgatagtaaccttttacccaggttttcagatgtgtttggaggagttttctgtcttctgagggctggtcctctttccttttcagcgtttactUpstream primer (SEQ ID NO: 35):F: 5′-gaatacctttaagaaggagatatacatgatcatgtggaatag gtgcgaggcag-3′Downstream primer (SEQ ID NO: 36):R: 5′-gaaccgtgacagtaaacgctgaaaaggaaagaggaccag-3′

(2). Design the primers and related sequences of the desired conversionregion. Human DNA fragment 324 bp (SEQ ID NO: 37). As compared to thenucleotide sequence from positions 5457087 to 5457410 of the NCBIaccession number NC_000009.12, the following nucleotide is different:87C→T, without affecting protein expression.

(SEQ ID NO: 37) gtcacggttcccaaggacctatatgtggtagagtatggtagcaatatgacaattgaatgcaaattcccagtagaaaaacaattagaTctggctgcactaattgtctattgggaaatggaggataagaacattattcaatttgtgcatggagaggaagacctgaaggttcagcatagtagctacagacagagggcccggctgttgaaggaccagactccctgggaaatgctgcacttcagatcacagatgtgaaattgcaggatgcaggggtgtaccgctgcatgatcagctatggtggtgccgactacaagcgaattactgtg

The PCR was done in two pieces:

For the first piece:

The upstream primer (SEQ ID NO: 38) is:

F: 5′-ctctttccttttcagcgtttactgtcacggttcccaaggacc tatatgtgg-3′

The downstream primer (SEQ ID NO: 39) is:

R: 5′-cccaatagacaattagtgcagccagatctaattgtttttcta ctgggaatttgc-3′

For the second pieces:

The upstream primer (SEQ ID NO: 40) is:

F: 5′-caattagatctggctgcactaattgtctattggga-3′

The downstream primer (SEQ ID NO: 41) is:

R: 5′-cttaccattgactttcacagtaattcgcttgtagtcggcacc a-3′

(3). Design the upstream primers of the homologous recombinationfragment and the downstream primers matching therewith, as well as otherrelated sequences.

Specifically:

3′ homologous arm (SEQ ID NO: 42), which was the nucleotide sequencefrom positions 29373890 to 29375042 of the NCBI accession numberNC_000085.6:

aaagtcaatggtaagaattaccctggatggggaaggcttcatccgtatttaaaacagctccctaatgttgagagctcttcattcttgagagttcgcacgcacttctcacagaacaacagcagcctgttcttctcgctcgtttgttcattcgttcgttcacacacttcaccagtgaaaaagcctagcactgtgtgtttgatagtaacttgagattcagtaccagataatactcagccatgctttgcagtcagtaccatgatcttgcaaaggtgaaatgccaggtgtttgtttcttatcataaatgcaatatataatatattacatagatgtatagatataactgtgtaacatgcaataagatataatatgcatatatttcatataacataatgtataatatataatgtataataatatatactacaatatatagttatatgcatagttatatattgcatttatgataaaaagcaaacacctggcatttcacttttgcaagctttttgaattacttgtaaatatatatacatgcaaacatacatacacacacatgtttttttacaagtaatttgaatgtcatggaaagaaatagaatcataaaaatgtccctcctccctaactaccatcttctaagcataaatatacagtaactactatttgtacatccctccatgactttttgattggattactgtttatatttaatctatcaggcttagcacattttctttcctttgaatacctccatacaaaattcaatgtgtgtttatatatatatgtatatatatagttatatcatatcatatatcatacaaagttttatatatgtatacatatataaacacacatatctacacatacatacacattttttatatatatacacaatatataatgtatatgtgtgtgtgtgtgcatatacctctatatctatctatctatctatctatctatctatctatctatctatctatctatagcttctactgtaagggtcactttttaaaaaattaaggttaatctatgaaggatgagaagtgaagatcttaagtgtagaagaagccgttcttccacagagatggtacaggctacactcagcaggcatgcattcattttcagggcctgcatctctgggagtgctgaggaggaact aUpstream primer (SEQ ID NO: 43):F: 5′-gcgaattactgtgaaagtcaatggtaagaattaccctggat gggg-3′Downstream primer (SEQ ID NO: 44):R: 5′-gcgtcggttgttagcagccggatctcagtagttcctcctcag cactcccagag-3′

C57BL/6 mouse DNA or BAC library is used as the template to carry outPCR amplification for exon 3's 5′-terminal homologous arm fragment (SEQID NO: 34) and 3′-terminal homologous arm fragment (SEQ ID NO: 42).Human DNA is used as the template to carry out PCR amplification for thehuman DNA fragment (SEQ ID NO: 37), and the AIO kit is used to ligatethe fragments to the pClon-4G plasmid provided by the kit, so as toobtain the vector pClon-4G-PD-L1.

Example 3. Verification of Vector pClon-4G-PD-L1

Two pClon-4G-PD-L1 clones were randomly selected and identified by threesets of enzymes. Among them, NcoI should generate 3615 bp+2551 bp+439 bpfragments, BglII+NdeI should generate 3775 bp+1553 bp+1282 bp fragments,HindIII should generate 4108 bp+2354 bp+143 bp fragments. The resultsfor Plasmids 1 and 2 were in line with the expectations (FIG. 6). Thesequences of Plasmids 1 and 2 were verified by sequencing. Plasmid 2 wasselected for subsequent experiments.

Example 4. Microinjection and Embryo Transfer

The pre-mixed Cas9 mRNA, pClon-4G-PD-L1 plasmid and in vitrotranscription products of pT7-sgRNA-PDL1, pT7-sgRNA-PDL11 plasmids wereinjected into the cytoplasm or nucleus of mouse fertilized eggs (C57BL/6background) with a microinjection instrument (using in vitrotranscription kit to carry out the transcription according to the methodprovided in the product instruction). The embryo microinjection wascarried out according to the method described, e.g., in A. Nagy, et al.,“Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),”Cold Spring Harbor Laboratory Press, 2003. The injected fertilized eggswere then transferred to a culture medium for a short time culture, andthen was transplanted into the oviduct of the recipient mouse to producethe genetically modified humanized mice. The mice population was furtherexpanded by cross-mating and self-mating to establish stable mouselines. The humanized mouse obtained was named B-hPD-L1.

Example 5. Identification of Genetically Modified Humanized Mouse Model

1. Genotype Detection

PCR analysis was performed for mouse tail genomic DNA of 11 mice. Theprimers target exon 3 of the PD-L1 gene. The primers for PCR-1 werelocated on the left side of the 5′ homologous arm, primers for PCR-3were located on the right side of the 3′ homologous arm; in addition,primers for PCR-2 and PCR-3 were located on the humanized fragment. Theprimer sequences are shown below:

5′ terminus primers: PCR-1 (SEQ ID NO: 45):5′-TGGAAGAATGGCTCCTGTTTCCCAC-3′; PCR-2 (SEQ ID NO: 46):5′-CACCCCTGCATCCTGCAATTTCACA-3′ 3′ terminus primers:PCR-3 (SEQ ID NO: 47): 5′-ATTAGATCTGGCTGCACTAATTGTC-3′;PCR-4 (SEQ ID NO: 48): 5′-ATGAGTGAAGCTCTCAGGTCTATGC-3′

If the recombinant vector has the correct insertion, there should beonly one PCR band. The length of the 5′ terminus product should be 2120bp, and the length of the 3′ terminus product should be 1550 bp.

PCR Amplification Conditions:

TABLE 5 The PCR reaction system (20 μL) 10 × buffer 2 μL dNTP (2 mM) 2μL MgSO₄ (25 mM) 0.8 μL Upstream primer (10 μM) 0.6 μL Downstream primer(10 μM) 0.6 μL Mouse tail gDNA 200 ng KOD-Plus- (1 U/μL) 0.6 μL

TABLE 6 The PCR reaction conditions Temperature Time Cycles 94° C. 5 min1 94° C. 30 sec 15 67° C. (−0.7° C./cycle) 30 sec 68° C. 1 kb/min 94° C.30 sec 25 56° C. 30 sec 68° C. 1 kb/min 68° C. 10 min 1  4° C. 10 min 1

Among the 11 mice, 5 of them were identified as positive mice. Theidentification number for these mice are 4, 5, 7, 8, and 9. Theidentification results are provided in FIG. 7.

Furthermore, these 5 positive mice were examined by Southern blotting todetermine whether they had random insertions. The genomic DNA wasextracted from the mouse tail, and BhlII, PstI were used to digest thegenomic DNA. The digestion products were transferred to membrane andhybridized. The probes P1 and P2 were located respectively on the 5′homologous arm and outside the 3′ homologous arm. The primers for probesynthesis are as follows:

P1-F (SEQ ID NO: 49): 5′-ATCATGTGGAATAGGTGCGAGGCAG-3′P1-R (SEQ ID NO: 50): 5′-GAAAGAGCAGACGAGCCAGCTGTAA-3′P2-F (SEQ ID NO: 51): 5′-CAGACTAACACTCACTCCCTGCTGC-3′P2-R (SEQ ID NO: 52): 5′-AAACATCATTCGCTGTGGCGTTGAC-3′

The genetically engineered mice should have the 4.1 kb or 15.1 kb bandwith probe hybridization; whereas the wild type C56BL/6 mice would havethe 5.4 kb or 8.8 kb band, and no hybrid band should be generated.

The results showed that the bands were consistent with the expectedresults. It was confirmed that the 2 mice were positive hybrids that didnot have random insertions. They were the mice with identificationnumbers 4 and 5. Southern blot results are shown in FIG. 8.

It thus shows that this method can be used to construct humanizedB-hPD-L1 mice that have no random insertion.

2. Protein Identification

One of the humanized F1 generation mice identified by PCR was selectedfor the study. One wild type C57BL/6 mouse was used as the control. 15μg of CD3 were injected intraperitoneally to the mice, and in 24 h 15 μgof CD3 were further injected intraperitoneally to the mice. The spleenswere collected at the end of 39 h, and the spleen samples were grinded.The ground samples were then passed through 70 μm cell mesh, thefiltered cell suspensions were centrifuged and the supernatants werediscarded; the erythrocyte lysis solution was added for lysis of 5 min,and then PBS solution was added to neutralize the lysis reaction. Thesolution was centrifuged again and the supernatants were discarded. Thecells were washed once with PBS. The antibody staining was performed for30 to 45 min in darkness; and the cells were washed once again with PBS.Flow cytometry was carried out to detect protein expression. Flowcytometry analysis results (FIGS. 9A-9E) show that when compared withthe C57BL/6 mice with or without the stimulation of CD3 antibody for Tcell activation in spleen, the humanized mouse spleen had the cells ofhuman PD-L1 protein expression as detected by fluorescent anti-humanPD-L1 antibody, while the spleen of the C57BL/6 control mice did nothave detectable cells of human PD-L1 protein expression. The foregoingresults indicate that the genetically modified, humanized PD-L1 mouse isable to express human PD-L1 protein, which can be detected by ananti-human antibody. The model mice will be useful for screening anddetection of anti-human PD-L1 antibodies.

The B-hPD-L1 humanized genetically engineered homozygous mice wereobtained by mating the previously obtained heterozygous mice with eachother. One homozygous B-hPD-L1 mouse (4-6 weeks old) was selected, andtwo wild type C57BL/6 mouse were selected as a control. 7.5 μg of mouseCD3 antibody was injected intraperitoneally to the mice, and the spleensof the mice were collected after 24 h. The spleen samples were groundand then filtered through a 70 μm cell filter, the obtained cellsuspensions were centrifuged and the resulting supernatants werediscarded. The cell samples were added with erythrocyte lysis solutionfor lysis of 5 min, and then added PBS solution to neutralize the lysisreaction, centrifuged again and the supernatants were discarded, thecells were washed once with PBS. The obtained samples were used in FACSdetection and RT-PCR detection.

FACS detection: The T cells extracellular proteins were simultaneouslystained with mouse PD-L1 antibody mPD-L1 APC and mouse T cell surfaceantibody mTcRβ, as well as human PD-L1 antibody hPD-L1PE and mouse Tcell surface antibody mTcRβ; the cells were then washed with PBS andthen detected for protein expression by FACS detection. Flow cytometryanalysis results are shown in FIGS. 10A-10F, when compared with theC57BL/6 mice with or without the stimulation of CD3 antibody for T cellactivation in spleen, the mouse PD-L1 antibody was able to detect thecells expressing mouse PD-L1 protein in the spleen samples from theC57BL/6 control mice (FIG. 10B); while the mouse PD-L1 antibody wasunable to detect the cells expressing mouse PD-L1 protein in the spleensamples from B-hPD-L1 homozygote (FIG. 10C). Moreover, the human PD-L1antibody was able to detect the cells expressing human PD-L1 protein inthe spleen samples from B-hPD-L1 homozygote (FIG. 10F); while the humanPD-L1 antibody was unable to detect the cells expressing human PD-L1protein in the spleen samples from the C57BL/6 control mice (FIG. 10E).

RT-PCR detection: total RNA was extracted from the spleen cells ofB-hPD-L1 homozygotes, and cDNAs were then obtained by reversetranscription using a reverse transcription kit.

Primers for mPD-L1 RT-PCR: mPD-L1 RT-PCR F2: (SEQ ID NO: 53)5′-CTGGACCTGCTTGCGTTAGT-3′, and mPD-L1 RT-PCR R2: (SEQ ID NO: 54)5′-CGTCTGTGATCTGAAGGGCA-3′were used to amplify mouse PD-L1 fragment of 169 bp.

Primers for hPD-L1 RT-PCR: hPD-L1 RT-PCR F2: (SEQ ID NO: 55)5′-TTAGATCTGGCTGCACTAAT-3′, and hPD-L1 RT-PCR R2: (SEQ ID NO: 56)5′-AGTGCAGCATTTCCCAGGGA-3′were used amplify human PD-L1 fragment of 155 bp.

PCR reaction system is 20 reaction conditions: 95° C., 5 min; (95° C.,30 sec; 60° C., 30 sec; 72° C., 30 sec, 35 cycles); 72° C., 10 min; and4° C. GAPDH was used as an internal reference.

The results are shown in FIG. 11. The mRNA expression of mouse PD-L1 canbe detected in the activated cells of wild-type C57BL/6 mice; while themRNA expression of human PD-L1 can be detected in the activated cells ofthe B-hPD-L1 homozygous mouse.

Example 6. Identification of Gene Knockout Mice

Since the cleavage of Cas9 results in DNA double strands break, and thehomologous recombination repair may result in insertion/deletionmutations, it is possible to obtain PD-L1 knockout mouse while preparingthe humanized PD-L1 mouse. A pair of primers was thus designed. They arelocated on the left side of the 5′ end target site, and to the rightside of the 3′ end target site, which are shown as follows:

5′-gcatcaagcttggtaccgataggtgcaatcccaacaccagaaaca-3′ (SEQ ID NO: 57)

5′-acttaatcgtggaggatgatagtgcgtgcgaactctcaagaatga-3′ (SEQ ID NO: 58)

The PRC reaction systems and conditions are listed in Table 7 and Table8. Under this condition, the wild type mice should have only one PCRband at the length of 535 bp; and the homozygotes should have only onePCR band. The results are shown in FIG. 12. The mice with identificationnumbers EL15-76, EL15-77, EL15-78 are PD-L1 knockout mice.

TABLE 7 The PCR reaction system (20 μL) 2 × Master Mix 10 μL Upstreamprimer (10 μM) 0.5 μL Downstream primer (10 μM) 0.5 μL Mouse tailgenomic DNA 200 ng KOD-Plus- (1 U/μL) 0.6 μL ddH₂O Add to 20 μL

TABLE 8 The PCR reaction conditions Temperature Time Cycles 95° C. 5 min1 95° C. 30 sec 30 59° C. 30 sec 72° C. 30 sec 72° C. 10 min 1  4° C. 10min 1

Example 7. Anti-Human PD-L1 Antibody In Vivo Efficacy Verification

This example selects Atezolizumb (commercial name Tecentriq), the firstapproved human PD-L1 antibody, to evaluate the in vivo efficacy in ahumanized animal model. This drug was developed by Genentech of Roche.It was approved in May 2016. Its primary indications are for thetreatment of the patients with locally advanced or metastatic urothelialcarcinoma whose conditions were still in progress in the 12-month periodof, before or after the treatment of platinum-based chemotherapy orreceiving a new or adjuvant platinum chemotherapy, as well as the secondline treatment for non-small cell lung cancer (NSCLC). There arecurrently a number of other indications in the clinical trial stage,including breast cancer, bladder cancer, prostate cancer and so on.

Mice containing humanized PD-L1 gene (for example, the B-hPD-L1 miceprepared by the methods described herein) were injected subcutaneouslyon right body side with 5×10⁵ mice colon cancer cell MC38. When thetumor volume has reached about 100 mm³, the mice were randomly dividedinto control group and treatment group (n=5 animals/group). The controlgroup (G1) was injected with blank solvent in an equal volume. Thetreatment group (G2) was intraperitoneally injected with 1-10 mg/kg ofanti-human PD-L1 antibody Atezolizumb, the injections were administeredevery other day, totally eight times of injection. The tumor volume wasmeasured twice a week and the body weight was measured for each mouse.Moreover, euthanasia should be performed when the tumor volume of asingle mouse reached 3000 mm³.

Overall, the animals in each group were healthy, and the body weights ofall the treatment and control mice did not significantly changethroughout the experimental period (FIG. 13). In FIG. 14, the tumor inthe control group continued growing during the experimental period; whencompared with the control group mice, the tumor volumes in the treatmentgroup were smaller by a certain degree (FIG. 14). It thus can bedetermined that the use of anti-human PD-L1 antibody Atezolizumbsignificantly inhibited the tumor growth in mice.

Table 9 shows the tumor volumes on the day of grouping, 10 days afterthe grouping, and at the end of experiment (17 days after the grouping),the survival rate of the mice, the tumor (volume) inhibition rate (TumorGrowth Inhibition Value, TGI_(TV)), and the statistical differences (Pvalue) in mouse body weights and tumor volume between the treatment andcontrol groups.

TABLE 9 Tumor volume (mm³) P value Day 0 Day 10 Day 17 Survival No TumorTGI_(TV) % Tumor volume Control 115 ± 26 1022 ± 536 1653 ± 963  5/5 0/5N/A N/A Treatment 114 ± 28 172 ± 54 58 ± 83 5/5 3/5 103.1 0.026 (10mg/kg) Treatment 114 ± 27  406 ± 471 548 ± 858 5/5 2/5 71.8 0.092 (3mg/kg) Treatment 113 ± 26  268 ± 123 442 ± 296 5/5 0/5 78.6 0.028 (1mg/kg)

Table 9 shows that all animals in both treatment and control group werealive at the end of the experiments (17 days after the grouping). Basedon FIG. 13, the weight of the animals, and the weight change over theexperimental period did not have much difference between the treatmentgroup and the control group. The tumor in the control group continuedgrowing during the experimental period; while in the treatment group of15 mice, 5 mice had no tumor at the end of the experiment. At the end ofthe experiment, the average tumor volume in the control group is1653±963 mm³; and the average tumor volume in the treatment groups are58±83 mm³, 548±858 mm³, and 442±296 mm³, at the doses of 10 mg/kg, 3mg/kg, and 1 mg/kg respectively. The tumor volumes in the treatmentgroups were smaller than the control group, with TGI_(F) being 103.6%,71.8%, and 78.6%, indicating that different doses of anti-human PD-L1antibody Atezolizumb has significant inhibitory effect on tumors(TGI_(TV)>60%). The effects are better with higher doses. This examplehas demonstrated that the anti-human PD-L1 antibody Aezolizumb showsrelatively strong inhibitory effects on tumor growth in B-hPD-L1 mice,is relatively safe, and well tolerated by the animals.

Example 8. Preparation and Identification of Mice with Double Humanizedor Multiple Humanized Genes

Mice with the humanized PD-L1 gene (such as the B-hPD-L1 animal modelprepared using the methods as described in the present disclosure) canalso be used to prepare a double-humanized or multi-humanized animalmodel. For example, in Example 4, the fertilized egg cells used in themicroinjection and embryo transfer process can be selected from thefertilized egg cells of other genetically modified mice or thefertilized egg cells of B-hPD-L1 mice, so as to obtain PD-L1 humanizedand other gene modified double or multiple gene modified mouse models.

In addition, the B-hPD-L1 animal model homozygote or heterozygote can bemated with other genetically modified homozygous or heterozygous animalmodels, and the progeny is then screened; according to the Mendelianlaw, there is a chance to obtain the PD-L1 humanized and other genemodified double genes or multiple genes modified heterozygous animalmodels, and then the obtained heterozygous can be mated with each otherto finally obtain the double genes or multiple genes modifiedhomozygote.

In the case of the generating double humanized PD-L1/PD-1 mouse, sincethe mouse PD-L1 gene and Pd-1 gene are located on different chromosomes,the double humanized PD-L1/PD-1 mouse was obtained by mating theB-hPD-L1 heterozygous mouse with B-hPD-1 homozygous mouse.

PCR analysis was performed on the mouse tail genomic DNA of doublehumanized PD-L1/PD-1 mice using four pairs of primers. The specificsequences and product lengths are shown in Table 10. The reaction systemand reaction conditions are shown in Table 7 and Table 8. The resultsfor a number of humanized PD-L1/PD-1 mice are shown in FIGS. 15A-15D,wherein FIGS. 15A and 15B show that the mice numbered 1-16 werehumanized PD-L1 homozygous mice; and FIGS. 15C and 15D show that themice numbered 1-16 were humanized PD-1 homozygous mice. The results ofthe two groups indicate that the 16 mice of the numbers 1-16 were doublegene homozygotes.

TABLE 10 Primer sequences Product Primer Sequence length PD-L1 MUTF: 5′-ccagggaggtggcccactg Mut: ataata-3′ (SEQ ID NO: 59) 528 bpR: 5′-cacccctgcatcctgcaat ttcaca-3′ (SEQ ID NO: 46) PD-L1 WTF: 5′-ccagggaggtggcccactg WT: ataata-3′ (SEQ ID NO: 59) 356 bpR: 5′-actaacgcaagcaggtcca gctccc-3′ (SEQ ID NO: 60) PD-1 MUTF: 5′-cttccacatgagcgtggtc Mut: agggcc-3′ (SEQ ID NO: 61) 325 bpR: 5′-ccaagggactattttagat gggcag-3′ (SEQ ID NO: 62) PD-1 WTF: 5′-gaagctacaagctcctagg WT: taggggg-3′ (SEQ ID NO: 63) 345 bpR: 5′-acgggttggctcaaaccat taca-3′ (SEQ ID NO: 64)

The expression of the double humanized PD-L1/PD-1 mice was furtherexamined. A double humanized PD-L1/PD-1 homozygote (6 weeks old) wasselected for the study. Two wild type C57BL/6 mice were selected ascontrol. Mice were injected with 7.5 μg of mouse CD3 antibodyintraperitoneally. After 24 hours, the mice were euthanized, and thenthe spleens of the mice were collected. The spleen samples were groundand the ground samples were filtered through a 70 μm cell mesh. Thefiltered cell suspensions were centrifuged and the supernatants werediscarded; erythrocyte lysis solution was added for lysis for 5 min, andthen PBS solution was added to neutralize the lysis reaction. FACS andRT-PCR analysis were then performed.

FACS detection: The T cells extracellular proteins were simultaneouslystained with mouse T cell surface antibody mTcRβ and mouse PD-L1antibody mPD-L1 APC (FIGS. 16A, 16B, 16C), human PD-L1 antibody hPD-L1PE (FIGS. 16D, 16E, 16F), mouse PD-1 antibody mPD-1 PE (FIGS. 17A, 17B,17C), or human PD-1 antibody hPD-1FITC (FIGS. 17D, 17E, 17F). Thesamples were washed with PBS and analyzed by FACS. Flow cytometryanalysis results are shown in FIGS. 16A-16F, 17A-17F. When compared withthe C57BL/6 mice with or without the stimulation of CD3 antibody for Tcell activation in spleen, the human PD-L1 antibody and human PD-1antibody were able to detect the cells expressing human PD-L1 and PD-1in the spleen samples from the humanized PD-L1/PD-1 homozygotes; whileno such detection was observed in control spleen samples from C57BL/6control mice.

RT-PCR detection: total RNA was extracted from the spleen cells of wildtype C57BL/6 mice and humanized PD-1/PD-L1 homozygotes, and the cDNAwere then obtained by reverse transcription using a reversetranscription kit.

The primers mPD-L1 RT-PCR F2 (SEQ ID NO: 53) and mPD-L1 RT-PCR Rc (SEQID NO: 54) were used to amplify mouse PD-L1 fragment of 169 bp.

The primers hPD-L1 RT-PCR F2 (SEQ ID NO:55), and hPD-L1 RT-PCR R2 (SEQID NO:56) were used to amplify human PD-L1 fragment of 155 bp.

The primers mPD-1 RT-PCR F3: 5′-CCTGGCTCACAGTGTCAGAG-3′ (SEQ ID NO:65)and mPD-1 RT-PCR R3: 5′-CAGGGCTCTCCTCGATTTTT-3′ (SEQ ID NO:66) were usedto amplify mouse PD-1 fragment of 297 bp.

The primers hPD-1 RT-PCR F3: 5′-CCCTGCTCGTGGTGACCGAA-3′ (SEQ ID NO:67),and hPD-1 RT-PCR R3: 5′-GCAGGCTCTCTTTGATCTGC-3′(SEQ ID NO:68) were usedto amplify human PD-1 fragment of 297 bp.

PCR reaction system was 20 μL, reaction conditions: 95° C., 5 min; (95°C., 30 sec; 60° C., 30 sec; 72° C., 30 sec, 35 cycles); 72° C., 10 min;and 4° C. GAPDH was used as an internal reference.

The results are shown in FIGS. 18 and 19. The mRNA expression of mousePD-L1 and PD-1 could be detected in the activated cells of wild-typeC57BL/6 mice; while the mRNA expression of human PD-L1 and PD-1 could bedetected in the activated cells of the PD-1/PD-L1 homotygotes mice.

Example 9. Preparation Method Based on Embryonic Stem Cells

The non-human mammals can also be prepared through other gene editingsystems and approaches, which includes, but is not limited to, genehomologous recombination techniques based on embryonic stem cells (ES),zinc finger nuclease (ZFN) techniques, transcriptional activator-likeeffector factor nuclease (TALEN) technique, homing endonuclease(megakable base ribozyme), or other molecular biology techniques. Inthis example, the conventional ES cell gene homologous recombinationtechnique is used as an example to describe how to obtain a PD-L1 genehumanized mouse by other methods. According to the gene editing strategyof the methods described herein and the humanized mouse PD-L1 gene map(FIG. 4), a targeting strategy has been developed as shown in FIG. 20.FIG. 20 shows the design of the recombinant vector. In view of the factthat one of the objects is to replace the exons 1-5 of the mouse PD-L1gene in whole or in part with the human PD-L1 gene fragment, arecombinant vector that contains a 5′ homologous arm (4208 bp), a 3′homologous arm (5113 bp) and a humanized gene fragment (324 bp) is alsodesigned. The vector can also contain a resistance gene for positiveclone screening, such as neomycin phosphotransferase coding sequenceNeo. On both sides of the resistance gene, two site-specificrecombination systems in the same orientation, such as Frt or LoxP, canbe added. Furthermore, a coding gene with a negative screening marker,such as the diphtheria toxin A subunit coding gene (DTA), can beconstructed downstream of the recombinant vector 3′ homologous arm.Vector construction can be carried out using methods known in the art,such as enzyme digestion and so on. The recombinant vector with correctsequence can be next transfected into mouse embryonic stem cells, suchas C57BL/6 mouse embryonic stem cells, and then the recombinant vectorcan be screened by positive clone screening gene. The cells transfectedwith the recombinant vector are next screened by using the positiveclone marker gene, and Southern Blot technique can be used for DNArecombination identification. For the selected correct positive clones,the positive clonal cells (black mice) are injected into the isolatedblastocysts (white mice) by microinjection according to the methoddescribed in the book A. Nagy, et al., “Manipulating the Mouse Embryo: ALaboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press,2003. The resulting chimeric blastocysts formed following the injectionare transferred to the culture medium for a short time culture and thentransplanted into the fallopian tubes of the recipient mice (white mice)to produce F0 generation chimeric mice (black and white). The F0generation chimeric mice with correct gene recombination are thenselected by extracting the mouse tail genome and detecting by PCR forsubsequent breeding and identification. The F1 generation mice areobtained by mating the F0 generation chimeric mice with wild type mice.Stable gene recombination positive F1 heterozygous mice are selected byextracting rat tail genome and PCR detection. Next, the F1 heterozygousmice are mated to each other to obtain genetically recombinant positiveF2 generation homozygous mice. In addition, the F1 heterozygous mice canalso be mated with Flp or Cre mice to remove the positive clonescreening marker gene (neo, etc.), and then the PD-L1 gene humanizedhomozygous mice can be obtained by mating these mice with each other.The methods of genotyping and phenotypic detection of the obtained F1heterozygous mice or F2 homozygous mice are similar to those used inExample 5 described above.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A genetically-modified mouse whose genomecomprises at least one chromosome comprising a nucleotide sequenceencoding a chimeric programmed death-ligand 1 (PD-L1) at an endogenousPD-L1 gene locus, wherein the chimeric PD-L1 comprises SEQ ID NO: 33,wherein the mouse expresses the chimeric PD-L1 on the surface of one ormore spleen cells after the genetically modified mouse is challenged byanti-CD3 antibody.
 2. The mouse of claim 1, wherein the sequenceencoding the chimeric PD-L1 is operably linked to an endogenousregulatory element at the endogenous PD-L1 gene locus in the at leastone chromosome.
 3. The mouse of claim 1, wherein the mouse does notexpress endogenous PD-L1.
 4. The mouse of claim 1, wherein the mouse ishomozygous with respect to the sequence encoding the chimeric PD-L1 atthe endogenous PD-L1 gene locus.
 5. The mouse of claim 1, wherein themouse whose genome further comprises a nucleotide sequence encoding ahumanized PD-1 protein at the endogenous PD-1 gene locus, wherein themouse expresses the humanized PD-1 protein on the surface of one or morespleen cell after the genetically modified mouse is challenged byanti-CD3 antibody.
 6. The mouse of claim 1, wherein exon 3 at theendogenous PD-L1 gene locus is modified by CRISPR with sgRNAs thattarget SEQ ID NO: 1 and SEQ ID NO:
 11. 7. A genetically modified mouseor a progeny thereof, wherein the genetically modified mouse is made bya method comprising the steps of: modifying genome of an embryo of amouse by CRISPR with sgRNAs that target SEQ ID NO: 1 and SEQ ID NO: 11to introduce a fragment of human PD-L1 gene, wherein an endogenous PD-L1gene locus in the genome of the embryo is modified; and transplantingthe embryo to a recipient mouse to produce the genetically-modifiedmouse, wherein the genetically-modified mouse expresses a chimeric PD-L1comprising a fragment of human PD-L1 protein on the surface of one ormore spleen cells after the genetically-modified mouse is challenged byanti-CD3 antibody.
 8. The genetically-modified mouse or the progenythereof of claim 7, wherein the mouse has a C57BL/6 background.
 9. Agenetically-modified mouse whose genome comprises at least onechromosome comprising a sequence encoding a human PD-L1 at an endogenousPD-L1 gene locus, wherein the sequence encoding the human PD-L1 isoperably linked to an endogenous regulatory element at the endogenousPD-L1 gene locus in the at least one chromosome, wherein the mouseexpresses a human PD-L1 on the surface of one or more spleen cells afterthe genetically modified mouse is challenged by anti-CD3 antibody. 10.The mouse of claim 9, wherein the mouse does not express endogenousPD-L1.
 11. The mouse of claim 9, wherein the mouse is homozygous withrespect to the sequence encoding the human PD-L1 at the endogenous PD-L1gene locus.